Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
DISINFECTION OF DORMANT TRANSPLANTS AND SEEDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0211711
Grant No.
(N/A)
Project No.
CA-D-PPA-7696-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2007
Project End Date
Sep 30, 2012
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
Non Technical Summary
Growers are advised to plant GCLclean,GCY i.e., pathogen-free, seeds and transplants. Here, we propose to develop methods to use peracetic acid in elevated but not phytotoxic temperatures to disinfect seed and dormant transplants. We will focus on disinfection of the fungus Colletotrichum acutatum and the bacterium Xanthomonas fragariae in strawberry transplants and on disinfection of vegetable seeds.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21211221160100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1122 - Strawberry;

Field Of Science
1160 - Pathology;
Goals / Objectives
We propose to use peracetic acid at elevated, but not phytotoxic temperatures, to disinfest and disinfect planting material. Our first objective is to determine the efficacy of peracetic acid for disinfection and disinfestation of strawberry plants naturally infected with the anthracnose-causing fungus Colletotrichum acutatum and the angular leaf spot-causing bacterium Xanthomonas fragariae. Our second objection is to determine the efficacy of peracetic acid for disinfection and disinfestation of selected seed-borne diseases in which improvement is needed in the currently available controls.
Project Methods
Application of a treatment between harvest by a seed or dormant-plant producer and planting by a fruit or vegetable farmer would fit into many current agricultural practices. Because the treatment would be applied in a contained environment, the environmental impact of the treatment can be minimized. In addition, we selected peracetic acid, which is approved for use in the food industry, and indeed is widely used, because it degrades into acetic acid; peracetic acid also is listed by the Organic Materials Research Institute as suitable for organic production. Nonetheless, peracetic acid is an extremely effective disinfectant, and is more effective in some applications than other disinfectants including bleach, chlorine dioxide and hydrogen peroxide. Based on preliminary work, we have selected case studies in which we postulate that peracetic acid at elevated but non-phytotoxic temperatures will be effective at disinfection of strawberry transplants and of vegetable seeds with selected bacterial and fungal pathogens. We will start with in vitro experiments in a thermocycler in which we will document the kill of the selected pathogens as a function of the time of exposure, concentration of peracetic acid, and temperature. Then we will conduct in vivo experiments to document the percentage kill in naturally infected plant material and to make certain that the treatments have no adverse impact on subsequent plant growth and productivity.

Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: Results were communicated to a large private nursery and to UCCE farm advisors. PARTICIPANTS: University of California staff: Dr. Sukhwinder Kaur, Staff Research Associate; Rebecca Kim, undergraduate; Alex Aquino, undergraduate, and William Goss, undergraduate Both undergraduates were trained in laboratory techniques, experimental design, and in data collection and evaluation. Partner orgarnizations: University of California Cooperative Extension Farm Advisors Daniel Marcum and Stephen Vasquez. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In California, grapevine nurseries maintain conditions that are unintentionally conducive for propagation and dissemination of pathogens; these pathogens might not become apparent in vineyards until some years after transplantation. For example, a "seasoning room" is typically operated at 100% relative humidity with sufficient air flow that air-borne pathogens would be circulated. In a trial run, we used an air sampler to detect Phaeoacremonium aleophilum and then used real-time polymerase chain reaction to quantify the fungus in the seasoning room. Based on macroscopic observations of grafted material, we postulated that there also was transmission of the fungi Botrytis cinerea and Fusarium proliferatum in the seasoning room. We conclude that greater pathogen monitoring within the facility, and adjustment of environmental conditions including utilization of an air disinfection system would produce a higher quality transplant.

Publications

  • No publications reported this period


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The goal of the project was to test new technologies that might be used to disinfect plant pathogens in dormant transplants and seeds. We were particularly interested in the addition of residue-free oxidizing agents, especially peracetic acid, in some cases with elevated temperature, that might also allow disinfestation and possibly disinfection without the risk of pathogen dissemination, and potentially without the phytotoxicity associated with hot water treatments. We were also interested two other alternatives: 1) a metabolic stress disinfection and disinfestation (MSDD), an alternation of low vacuum and high pressure that might either allow infusion of either fungicides or fungitoxic volatile compound but not phytotoxic compounds into seeds or dormant vegetative transplanting material; and 2) specific radiofrequencies, which might permit heating without the potential for the pathogen dissemination that can occur with hot water. Unfortunately, we had no breakthroughs. However, we documented two current agricultural practices that currently have unrecognized problems with pathogens. First, currently, the California Department of Food and Agriculture requires that grapevine nurseries control for vine mealybug with a hot water dip of all material that enters the nursery. We discovered an incident in which the fungal pathogen Botryosphaeria rhodina, which can be albeit uncommonly be present in pycnidia in cracks in dead wood on the canes, was disseminated onto the freshly cut canes and caused infection in apparently 100% of the canes in the bath. Although laboratory treatments that simulated the vine mealybug nursery treatment killed 70% of the B. rhodina conidia, 30% survived. As far as we know, the particular water bath was functioning at the required temperature. We proposed to the California Grape Rootstock Improvement Commission that they sponsor research that would validate the modification of the circulating hot water tanks with a system that would generate water vapor which would have the same heating potential but neither the risk of pathogen dissemination nor the energy costs of heating large volumes of water. However, they were not interested in a changed regulation. Secondly, we documented that the hot water treatment of dormant strawberry transplants for control of Colletotrichum acutatum was less effective than widely believed. We communicated this to the strawberry nurseries and a publication is in preparation. PARTICIPANTS: University of California staff: Dr. Sukhwinder Kaur, Staff Research Associate II; Rebeka Kim, undergraduate; Alex Aquino, undergraduate. Both undergraduates were trained in laboratory techniques, experimental design, and in data collection and evaluation. Partner orgarnizations: Dr. Manuel Lagunas-Solar, Professor Emeritus of University of California at Davis and currently at RF Biocidics. Some of the research was conducted RF Biocidics in Vacaville CA; the company is developing and commercializing two technologies to disinfect and disinfest pests and pathogens from agricultural food: specific radiofrequencies (RF) and a metabolic stress disinfection and disinfestation (MSDD) that combines short periods of low pressure (vacuum) and generally elevated CO2 with ethanol vapor to control arthropod pests and potentially pathogens on commodities. TARGET AUDIENCES: We interacted with a number of UCCE farm advisors, especially Daniel Marcum, Oleg Daugovish, Mark Bolda, and Steve Koike. We also interacted with a number of nurseries and commodity groups including the California Celery Research Advisory Board, the California Strawberry Commission, California American Viticulture Foundation, California Fresh Carrot Advisory Board, California Leafy Greens Research Program, California Grape Rootstock Improvement Commission, the California Table Grape Commission, Duarte Nursery, Crown Nursery, Lassen Canyon Nursery, and Driscoll Nursery. We also interacted with RF Biocidics. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We primarily proposed to use peracetic acid at elevated, but not phytotoxic temperatures, to disinfest and disinfect planting material. Our main objective was primarily to determine the efficacy of peracetic acid (PAA) for disinfection and disinfestation of strawberry plants that were "naturally infected" with the anthracnose-causing fungus Colletotrichum acutatum. Although preliminary results were promising, we confronted two issues. One, the standard temperature and times used in a hot water treatment to supposedly kill C. acutatum readily kill superficial conidia, but do not kill, at least efficiently, C. acutatum hyphae; some C. acutatum mycelia in root pieces survived 127 degrees F for 15 min. Second, in plants that we believe simulate natural infections, the hyphae extend deep into the cortical tissues where the hyphae appears to be well protected. Ultimately, we could not find any PAA concentration/temperature /time combination that achieved sufficient kill of C. acutatum in strawberry plants with "natural" infections with acceptable levels of phytotoxicity. Currently, several nurseries use hot water (typically 10 minutes at 120 F) at early generations in vegetative "mother" production as a curative for C. acutatum; the treatment is only used in early generations because it reduces vigor. In replicated trials with both cv. Camarosa and Ventana, we documented significant phytotoxicity with the hot water treatment, and no significant reduction in incidence of C. acutatum-infected plants in four of the five trials. In one trial with Camarosa, there was a significant (P=0.05) reduction in the incidence of C. acutatum infection, but 14% remained infected (in comparison to 67% in the untreated control). We also treated transplants in the fungicide Switch (at a rate of 5 oz of formulated cyprodinil and fludioxonil per 100 gal water) by either submersion for 15 min or for a 15 minute treatment in a "MSDD" evacuation-compression chamber designed to promote infusion of the fungicide; plants were then grown out in the greenhouse before assay for C. acutatum. There was no detectable effect (P=0.5) of either of Switch treatments with 52% infection in the untreated control (n=96), 48% infection in the Switch dip (n=96) and 42% infection in the Switch MSDD (n=52). In addition, we were unable to use radiofrequency to kill C. acutatum without killing the strawberry tissue. Similarly, our experiments using radiofrequency to kill the fungus Verticillium dahliae in spinach seed without significant decrease in seed germinability were unsuccessful. We also used a variety of concentrations of PAA at varying temperatures and times to cure carrot seed of Alternaria dauci, A. radicina and Xanthomonas campestris pv. carotae (Xcc). We conclude that PAA is not more effective than the current hot water treatment in treating carrot seeds against the fungal pathogens, and indeed the fungi provide routes by which PAA gains access to (and damages) the embryo. Our results are consistent with the hypotheses that Xcc is primarily localized in the seed coat, that Xcc infection does not disrupt the integrity of the seed coat, and that PAA can disinfect seed-borne Xcc.

Publications

  • No publications reported this period


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Results regarding the dissemination of Botryosphaeria and other grapevine pathogens in the hot water bath designed to control grapevine mealybug (Planococcus ficus) were presented at a meeting of the California Grape Rootstock Improvement Commission in June 2010. Results on disinfection of carrot seed were presented in a proposal to the California Leafy Greens Board. PARTICIPANTS: University of California staff: Dr. Sukhwinder Kaur, Staff Research Associate; Rebecca Kim, undergraduate; Alex Aquino, undergraduate. Both undergraduates were trained in laboratory techniques, experimental design, and in data collection and evaluation.<br> Partner orgarnizations: Dr. Manuel Lagunas-Solar, Biodidics and University of California at Davis; Dr. Lindsey DuToit, Washington State University, Mount Vernon NWREC; Dr. Krishna Subbarao, University of California at Davis and USDA-ARS TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In California, grapevine nurseries are required to treat cuttings with a hot water treatment at 52 C for 5 minutes to control the grapevine mealybug. We investigated an incident in which the hot water treatment had disseminated the fungal pathogen Botryosphaeria rhodina to all of the cuttings that had been in the same water bath. Our results indicated that a hot water treatment with a circulating water bath was conducive to dissemination of multiple fungal pathogens that, while reduced in population, survived the 5 minute exposure to 52 C. We conclude that a mist-generated heat treatment could be developed that would be equally efficacious for mealybug control, would ultimately have lower heating costs for nurseries, and would have less risk for dissemination of water-disseminated spores. Our second project concerns Verticillium-infected spinach seed in California; once fields are infested, it is extremely difficult to remove the pathogen. We used batches of spinach seed, obtained from Dr. Krishna Subbarao, in which 60% were naturally infected with Verticillium dahliae. In collaboration with Dr. Manuel Lagunas-Solar and the Biocidics company in Vacaville, California, we tested the effect of selected power and time of radiofrequency (RF) on disinfection. Although we reduced seed germination in all treatments in which we completely eliminated Verticillium dahliae, we consider the results extremely promising because we have an unexplored range of conditions (RF power and time) which are likely to be fungitoxic without phytotoxic. We note that our efficacious RF treatments were all relatively short, i.e., less than 7.5 minutes.

Publications

  • No publications reported this period


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Results were presented February 24, 2010 in Visalia California as a 30 minute invited presentation at the San Joaquin Valley Table Grape Seminar, which is sponsored by the California Table Grape Commission. The talk was entitled "A single pre-harvest peracetic acid treatment for postharvest disease control." Results were also presented as a poster at the Annual Meeting of the American Phytopathology Society, which was held in Portland, Oregon in June 2009. PARTICIPANTS: Participants included two cooperative extension farm advisors, one staff research associate, and three undergraduate students. Partner organizations included the California Table Grape Commission. TARGET AUDIENCES: Target audiences include table grape growers. Our cooperative extension farm advisor collaborator is active in extension and outreach. PROJECT MODIFICATIONS: Research is proceeding as planned.

Impacts
Here we report results on use of peracetic acid as a pre-season application in the field on post-harvest rot. Botrytis cinerea, causal agent of bunch rot, is a common pathogen of stored table grapes. Growers manage bunch rot with multiple fungicide applications in the field and sulfur dioxide during storage. Organic table grape growers have few in-season treatments available and cannot use sulfur dioxide for postharvest management. An organically approved, pre-harvest disinfestation that decreases post-harvest losses would be beneficial for organic and possibly for conventional table grape production. The disinfectant peracetic acid (PAA), which also contains acetic acid and hydrogen peroxide, is currently registered as a dip or spray on raw unprocessed fruit (with no restrictions on immediate consumption) at 85 ppm (0.0085%). Based on preliminary evidence, we applied 0.05% PAA one-day pre-harvest in 2008 and 2009. Cultivars included Thompson seedless (TS), Crimson Seedless (CS), Red Globe, and Princess. PAA significantly decreased rot in storage in some but not in all trials. For example, in the organic TS vineyard in 2008, rot after one month in storage was significantly reduced from 10.4% (fresh weight) in the untreated control to 4.3%; there were no significant differences between a PAA application one-day pre-harvest and an application the day of harvest. In both conventional TS vineyards in 2008, a field application of 0.05% PAA significantly reduced post-harvest rot. In a TS Madera vineyard in 2008, a one-day pre-harvest PAA application reduced rot after two months storage from 7.2% in the untreated control to 1.9%, and after three month storage, from 14.2 to 5.2%. Similarly, in the TS Fresno vineyard in 2008, a one-day pre-harvest PAA spray reduced rot after 37 days in storage from 8.7 to 2.2%. However, not all trials show a significant decrease in rot. We did not observe a significant effect of PAA in an organic trial on Princess, and in only one of two trials on conventionally-grown TS in 2009. Overall, PAA significantly decreased rot in only one of four trials of Crimson Seedless in which we could evaluate the data. When PAA was significantly effective, sulfur dioxide applied as a combination of gas and pads was generally significantly more so; efficacy of sulfur dioxide was so good that it was not possible to see if PAA improved efficacy of sulfur dioxide. Overall, the data suggest that some infections occur post-harvest and that PAA could be a feasible option for B. cinerea management, particularly in cases where sulfur dioxide is not used, or in cases where control in the presence of sulfur dioxide is less than desired.

Publications

  • Vasquez, S.. Epstein, L., Kaur, S, Holquin C. 2009. Peracetic acid treatment of fresh market grapes for post-harvest Botrytis cinerea control. Phytopathology 99:S134 (Abstr.)


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: This is a relatively new project and the results are not ready for dissemination. Reports have been submitted to the California Fresh Carrot Advisory Board the California Strawberry Commission. PARTICIPANTS: Participants included one graduate student, one undergraduate student, one visiting scientist, one staff research associate, and one cooperative extension farm advisor. Partner organizations included the California Fresh Carrot Advisory Board and the California Strawberry Commission. TARGET AUDIENCES: Target audiences include carrot seed producers, carrot producers, and strawberry nursery and strawberry fruit producers. Our cooperative extension farm advisor collaborator is active in extension and outreach. PROJECT MODIFICATIONS: Research is proceeding as planned.

Impacts
Financial resources are essential for conducting any research. We have focused on identifying combinations of temperatures and concentrations of peracetic acid that are toxic to selected pathogens, but are non-toxic to carrot seeds and strawberry daughter plants. We obtained carrot seed that was naturally infected with Xanthomonas campestris pv. carotae (Xcc); we confirmed that the pathogen was Xcc using polymerase chain reaction. After carrot seed was soaked in 0.1%, 0.3%, or 0.5% peracetic acid for ten minutes at either 68 degrees F, 104 degrees F or 113 degrees F, or soaked in water at 126 degrees F for 20 minutes, the seeds were air-dried and the percentage of seeds infected with Xcc was determined. The standard hot water treatment at 126 F for 20 minutes is relatively ineffective at controlling Xcc; Xcc incidence was significantly, but only reduced from 65% in the untreated seeds to 50% in the hot water-treated. In contrast, all of the peracetic acid treatments reduced Xcc incidence to 3% or less, without any significant reduction of seed germination. Because peracetic acid decomposes to acetic acid, oxygen, and water, we determined if it had to be rinsed off of the treated seed. The results indicate that it is critical to rinse off the 0.5% peracetic acid, particularly when it is treated at 113 degrees F, and that it becomes less important to rinse off the peracetic acid at lower concentrations and lower temperatures; including 0.3% peracetic acid at 104 degrees F. We have selected 0.3% peracetic acid at 104 degrees F for 10 minutes as the most promising treatment; increase of the peracetic acid concentration to 0.5% may start to have an inhibitory effect on seed germination. Elevation of the temperature to 113 degrees F does not appear to have any benefit. Because HCl is used to treat some bacterial diseases of tomato seeds, we conducted three trials in which the carrot seed was treated (unheated) in either 1.1 or 5% of concentrated HCl for either 10 or 30 minutes. Because these concentrations were relatively ineffective (albeit sometimes significantly) reducing Xcc incidence, we conducted two trials in which the seed was treated in 10% concentrated HCl for 10 or 30 minutes. While the 10% concentrated HCl for either 10 or 30 minutes was more efficacious in reducing Xcc than the standard hot water treatment for 20 minutes at 126 degrees F, the 0.3% peracetic acid for ten minutes was the most efficacious, and had no adverse effect on germination. In comparison to the untreated seed in which 79% germinated and 60% were infected with Xcc, the 0.3% peracetic acid treated seed at 104 degrees F had 89% germination and 0.6% with Xcc. Because our post-peracetic acid seeds still had a detectable incidence of Xcc (albeit generally less than 1%), we conducted two experiments with and without 0.01% of the silicon surfactant Breakthru in 0.3% peracetic acid at 104 degrees F for 10 min. The surfactant did not affect seed germination; the percentage of seed infected with Xcc was too low to see if the surfactant significantly improved the disinfection of Xcc or not.

Publications

  • No publications reported this period


Progress 01/01/07 to 12/31/07

Outputs
This is the first year of the project. Two undergraduate students and a visiting scientist were mentored on this project. Results were presented to the California Strawberry Commission.

Impacts
The disinfectant peracetic acid (PAA), also known as peroxyacetic acid, is a combination of acetic acid and hydrogen peroxide. PAA is often more effective than many other disinfectants, including bleach, chlorine dioxide, and hydrogen peroxide. It is approved for use in the food industry, and indeed is widely used, because it degrades into acetic acid. It also is listed by the Organic Materials Research Institute as suitable for organic production. Advantages of PAA include the following: the disinfectant properties of PAA have been well-studied; it is fast-acting; it is effective against many bacteria, fungi and nematodes; it is effective against biofilms; and it can be used in a broad temperature range. We have started testing PAA as a disinfectant of strawberry plants. Strawberry daughter plants can be both infected and infested with fungal and bacterial pathogens and nematodes. Currently, strawberry nurseries in California rely partly on a hot water treatment at 49 degrees C for 10 minutes in early generations of strawberry daughter production to disinfect mother plants of Colletotrichum acutatum, a fungal pathogen. However, in six trials, we have found that the hot water treatment is infective. We conducted a matrix of PAA concentration, time of exposure, and incubation temperature required to kill Colleotrichum acutatum in a variety of in vitro and in vivo assays. Not surprisingly, kill of mycelium in strawberry tissue required higher concentrations of PAA and/or exposure times than conidia. Higher incubation temperatures enhanced the effect of PAA. We selected 0.1% PAA and 0.05% PAA in 40 degrees C water for ten minutes as a condition with efficient kill of Colletotrichum acutatum but with neither observable phytotoxicity nor reduction of vigor of treated plants. In four independent trials, each with approximately 25 plants, 75% of the untreated plants were infected with Colletotrichum acutatum and those treated with hot water at 49 degrees C for 10 minutes had 78% infection. Plants treated in 40 degrees C water with 0.1% PAA and 0.05% PAA had significantly (P=0.05) less infection: 18% and 27%, respectively. In contrast, 78% of the plants treated in 40 degrees C water without PAA amendment were infected with Colletotrichum acutatum. No phytotoxicity was observed in plants treated with 40 degrees C water with 0.1% PAA and 0.05%. Additional experiments with strawberry plants infected with Colleotrichum acutatum, Xanthomonas fragariae, nematodes, or uninfected, and comparisons of growth of treated versus untreated plants are in progress.

Publications

  • No publications reported this period