CITRUS RESEARCH BOARD CRB Project No. 5600-103 PROJECT PLAN - RESEARCH GRANT PROPOSAL FOR FY2010-2011 Fiscal Year: 2010-2011 Anticipated Duration of Project: 5 Years This project is: New or X Ongoing (Year 3 of 5) Project Leader: J. E. Adaskaveg Location: Department of Plant Pathology, University of California, Riverside, CA 92521 Mailing address (if different) same as above Phone: 951-827-7577 or -3880 FAX: 951-827-7577 E-Mail: jim.adaskaveg@ucr.edu Cooperating Personnel: Project Title: Evaluation of New Postharvest Treatments to Reduce Postharvest Decays of Citrus Keywords: azoxystrobin, fludioxonil, pyrimethanil, fungicide resistance, management practices, blue and green mold Abstract (limit 200 words) New materials (synthetic, natural and biological) are being continuously evaluated for their potential use in managing postharvest decays of citrus. Our current focus is on new fungicides that are reduced-risk compounds. These materials are being evaluated, developed, and registered for postharvest use against blue and green mold caused by Penicillium spp. and sour rot caused by G. citri-aurantii. New materials such as fludioxonil (Graduate) and pyrimethanil (Penbotec) as well as the pre-mixtures fludioxonil-azoxystrobin (Graduate A+) and imazalil-pyrimethanil (Philabuster) have been registered and major usage is expected with approval of international tolerances (MRLs). Another material, propiconazole (Mentor), is being registered through IR-4 with efficacy against sour rot and imazalil-sensitive populations of P. digitatum. A pre-mixture of fludioxonil+azoxystrobin +propiconazole (Graduate TDC) is in development. Thus, this project is developing new materials and combinations of materials, developing baseline sensitivity data for future reference points in monitoring for resistance, and optimizing fungicide usage by identifying rates and application strategies (compatibility with fruit coatings, sanitizing agents, heat and ph-altering salts such as sodium bicarbonate) that maximize performance against Penicillium decays and sour rot. Furthermore, studies on the characterization of fungicide resistance are done including types (qualitative or quantitative), levels (low, medium, or high), fitness (pathogenic, non-pathogenic, and virulence), and frequencies of resistance in populations of P. digitatum to the new fungicides. Studies on the genetic diversity of G. citri-aurantii using mating type and molecular methods are being summarized for publication. Additionally, we have identified several potential fungicides that can be registered as preharvest or postharvest treatments for postharvest decay control of Penicillium and brown rot (Phytophthora) decays. Preharvest treatments would be applied immediately before harvest and may be especially important when fruit are stored for an extended time in field bins (e.g. degreening fruit, excessive fruit harvested) before postharvest treatments can be applied. Problem and its Significance*: New treatments for the management of postharvest decay are continuously being evaluated. Currently, several new postharvest fungicides have been registered for the citrus industry. This is mainly because of the *Use as much space as necessary; attach additional pages as needed; budget info and signatures will appear on last page. Not for publication without the express written consent of the project leader. Before quoting or reproducing any information in whole or extracted in any form, contact the project leader responsible.
widespread resistance in Penicillium populations against the older, still-registered fungicides and new, safer materials have recently become available. Azoxystrobin, fludioxonil, and pyrimethanil have all shown excellent control against Penicillium decays in our studies and all three materials have been classified as reduced risk fungicides by EPA. Syngenta, the manufacturer of azoxystrobin and fludioxonil, and Janssen Pharmaceutica, the registrant of pyrimethanil have obtained federal and state registrations for these fungicides. Advantages of newer fungicides include their lower residues for effective management of decays and their favorable safety characteristics. Another incentive for developing new materials of different classes is their use in fungicide resistance management programs. Examples of materials that have been studied by us include propiconazole (Mentor), tebuconazole (Elite), myclobutanil (Rally/Nova), tetraconazole (TM-415), CN- 2010A, iminoctadine tris-albesilate (TM-417), azoxystrobin (Abound), fludioxonil (Scholar), and pyrimethanil (Penbotec). The first five chemicals are triazoles - demethylation inhibitors similar to the widely used imazalil. CN-2010A is a new compound that will need further evaluation. Iminoctadine is a guanidinium; whereas the latter three are unrelated classes known as strobilurins or QoIs, phenylpyrroles, and anilinopyrimidines, respectively. Among the fungicides being registered, only propiconazole (Mentor) (and potentially CN- 2010A) has excellent activity against both, Penicillium decays and sour rot caused by Geotrichum citriaurantii. We have evaluated wax compatibilities of azoxystrobin, fludioxonil, pyrimethanil, and propiconazole and the results of these studies have directed fungicide labels and recommended application methods. We will continue to study staged or stepwise application of fungicides in aqueous and fruit coating (wax) applications on experimental packinglines to obtain optimum decay and sporulation control. We also will continue to evaluate the efficacy of azoxystrobin, fludioxonil, and pyrimethanil in single-fungicide and mixture treatments. To make applications with the new fungicides the most cost-effective, studies with re-circulating application systems such as flooder applications are ongoing. For this, proper sanitation practices that include sanitation of harvested fruit as well as sanitation of the re-circulating fungicide solution are most critical. Because chlorine is known to negatively interfere with the activity of imazalil and pyrimethanil, we will continue to evaluate alternatives including sodium bicarbonate and acidified hydrogen dioxide (registered names StorOx and Perasan or PAA) and food-use, quaternary ammonium products. Studies on the effect of these additives on fungicide efficacy, compatibility with waxes, and phytotoxicity potential are ongoing. Another focus of our research is the resistance potential to these new materials in target fungal populations. Because the fungicides belong to different fungicide groups, and thus, have different modes of actions against target organisms, they will be excellent in complementing imazalil in fungicide resistance programs. A general rule in resistance management is to rotate or mix different classes of pesticides because pesticides within one class have similar modes of action against target pest populations. Still, the potential for resistance may be high for these single-site mode-of-action materials. We are using the spiral gradient dilution method to estimate resistance potentials of fungicides. Results from our studies will ultimately help in providing guidelines for effective resistance management strategies in citrus packinghouses and help maintain a long-lasting efficacy of the new fungicides. Baseline sensitivities in Penicillium populations have been developed for the new fungicides fludioxonil, azoxystrobin, and pyrimethanil and are currently being developed for propiconazole using populations of Penicillium spp. and G. citri-aurantii. The SGD method allows us to evaluate many samples and large pathogen populations in a short period of time. Results from these studies will help to design usage patterns for the application of postharvest fungicides. Overall, our research objectives include new concepts for managing postharvest decays and reducing the development of resistance in populations of postharvest pathogens of citrus using integrated approaches. Currently, fungicide programs to reduce the risk of resistance from developing in a target populations include: 1) Maintain high labeled rates of single-site mode of action fungicides; 2) Limit the total number of applications of each class of fungicide to one or less for any lot of fruit; 3) Rotate between different classes of fungicides; 4) Initiate a fungicide disease management program with broad-spectrum, multi-site mode of action fungicides that reduce total population that subsequent single-site mode-of-action fungicides night encounter; and 5) Evaluate multi-site preharvest fungicides as protective treatments for reducing postharvest decays.
Objectives*: I. Continue to evaluate azoxystrobin, fludioxonil, pyrimethanil, propiconazole, pre-mixtures, and other new treatments for control of green mold and sour rot of citrus. A. Evaluate application methods and determine optimum rates for these fungicides and other treatments (e.g., CN-2010A) as single-treatments or as mixtures for optimum decay and sporulation control. Continue to evaluate the interaction of postharvest fungicides with sanitizing agents (e.g., chlorine, acidified hydrogen dioxide, quaternary ammonium products) or with phaltering materials (e.g., sodium bicarbonate). B. Continue to evaluate newly identified fungicides (e. g., chlorothalonil, azoxystrobindifenoconazole, i.e., Quadris Top) as preharvest treatments for postharvest decay control. C. Finalize data on efficacy and baseline sensitivities for propiconazole against P. digitatum and G. citri-aurantii and prepare manuscripts for publication. II. Evaluate new treatments (mandipropamid, fluopicolide, potassium phosphate - phosphorous acid) for control of brown rot caused by Phytophthora spp. A. Pre-harvest treatments for control of natural incidence of decay and after inoculation B. Postharvest treatments selected times after inoculation with Phytophthora sp. III. Continue to study fungicide resistance frequencies and mechanisms. A. Determine the competitiveness of less-sensitive isolates in co-inoculation studies. B. Continue to characterize fungicide resistance mechanisms in Penicillium spp. 1. Evaluate mutations in the cytochrome P-450 gene, the target of DMI fungicides, among DMI-resistant isolates. IV. Develop molecular identification methods for Penicillium spp. on citrus in California and for characterizing populations of G. citri-aurantii. A. Analyses of AFLP data obtained for isolates of G. citri-aurantii using numerical, non-linear statistical methods. B. Continue to develop PCR-based identification systems for postharvest pathogens. Project's Benefit to the Industry*: With registrations of azoxystrobin, fludioxonil, and pyrimethanil, and with all three potentially having international tolerances or maximum residue limits (MRLs) established in 2009 in many countries around the world, these materials will be quickly adopted and used extensively by the California citrus industry. Thus, with the future registration of propiconazole for postharvest use on citrus, these new fungicides represent the first major simultaneous fungicide introduction in the history of the citrus industry worldwide. These fungicides, representing different classes from each other, with the exception of propiconazole, from those previously registered, will be very important in developing future strategies in resistance management of imazalil- and thiabendazole-resistant populations of Penicillium species. Propiconazole represents the first highly effective fungicide ever registered for management of sour rot and is being developed and registered with the cooperation of IR-4 and the registrant. Guidelines are needed that will provide the industry with methods and usage strategies to optimize efficacy and reduce the risk for selection of resistant populations of pathogen species. New monitoring methods for fungicide resistance that we developed will provide fungicide users near-real time methods and allow for resistance strategies to be employed in a timely manner and natural resistance frequencies provide estimations of the fungicide resistance potential. Additionally, characterization of fungicide resistance mechanisms at the molecular base will provide a better understanding on the development of resistance. Usage recommendations included in the label that are based on our research will provide service companies and registrants the tools to develop effective decay and resistance management programs. Studies on the management of Phytophthora brown rot will provide new management options for this potentially destructive disease. Research Collaboration* (be specific): Collaboration is also being obtained from registrants of fungicides.
Plans and Procedures* (include site location and description in discussion of experimental design): I. Continue to evaluate azoxystrobin, fludioxonil, pyrimethanil, propiconazole, pre-mixtures, and other fungicide treatments for control of green mold and sour rot of citrus. A. Evaluate application methods and determine optimum rates for these fungicides as single-treatments or as mixtures for optimum decay and sporulation control. Continue to evaluate the interaction of postharvest fungicides with sanitizing agents (chlorine, sodium bicarbonate, acidified hydrogen dioxide). In these studies, fungicides will be applied to inoculated fruit either alone, in mixtures, or in combination with sanitizing agents (e.g., chlorine, acidified hydrogen dioxide, quaternary ammonium products) or with phaltering materials (e.g., sodium bicarbonate). For this, reduced fungicide concentrations (e.g., 200 ppm) will be used that are more discriminatory in showing any interaction effects. Applications will also be compared for different temperatures (e.g., between ca. 70 and 120F). The efficacy of a new DMI fungicide (CN-2010A) will be compared to that of imazalil and propiconazole. Fruit will be evaluated for incidence of decay and for phytotoxic effects. Fungicides will also be evaluated in experimental packingline studies using selected application methods. To make fungicide applications the most cost-effective a flooding application will be used where fungicide solutions can be sanitized and re-circulated. The staged application method with an aqueous fungicide application followed by a fungicide application in wax or by wax alone will be continued to be evaluated. Efficacy data will be used to develop labels with service companies and registrants allowing for aqueous dips or drenches (e.g., fungicides + sanitizers) and aqueous, storage wax, or pack wax applications of fungicides (e.g., fungicides without sanitizers) to fruit being stored or packed for marketing. Data will be evaluated using regression and analysis of variance procedures of SAS (Ver. 9.1). B. Evaluate newly identified fungicides as preharvest treatments for postharvest decay control of citrus. Recently, we have identified several fungicides that potentially can be registered as preharvest treatments that may provide some level of postharvest decay control as well as reduce total population of Penicillium spp. entering the packinghouse. These include chlorothalonil, azoxystrobin, difenoconazole (the premix azoxystrobin-difenoconazole), fenbuconazole, and another new DMI fungicide (CN-2010A). Treatments would be applied immediately before harvest and may be especially important for fruit that are stored for an extended time in field bins (e.g. de-greening Valencia orange fruit, excessive lemon fruit harvested) before postharvest treatments can be applied. Preharvest treatments will be applied at selected PHI intervals (e.g., 1 day and 7 day before harvest), fruit will be harvested and fruit will be stored under high humidity for 5 to 7 days at 20 to 25 C. Incidence of decay will be evaluated as a percentage of decayed fruit over total fruit harvest. C. Finalize data on efficacy and baseline sensitivities for propiconazole against P. digitatum and G. citriaurantii and prepare manuscripts for publication. Graduate student A. McKay is currently summarizing all his data and is preparing manuscripts for publication. Once published, reprints will be made available to those interested in the citrus industry. II. Evaluate new treatments (mandipropamid, fluopicolide, potassium phosphate - phosphorous acid) for control of brown rot caused by Phytophthora spp. In preharvest studies, applications at rates recommended by the manufacturers of the materials will be done to trees within 14 days of harvest. Fruit will be harvested (ca. 50 fruit/single-tree replication) and incubated for development of natural incidence of decay. Alternatively, harvested fruit will be inoculated with zoospores of P. citrophthora. Fruit will be incubated at 20C and evaluated after 8-10 days. In postharvest studies, fruit will be inoculated with zoospores and then diptreated after 12 to 18 h. Time studies (treatments at different times after inoculation) will be done with the most effective treatments. To potentially develop new fungicide pre-mixtures, the interaction between fungicides for brown rot control and newly registered postharvest fungicides (e.g., Graduate A+) will be tested to detect possible incompatibilities resulting in reduced performance.
III. Continue to study fungicide resistance frequencies and mechanisms. A. Determine the competitiveness of less-sensitive isolates of P. digitatum in co-inoculation studies. Mixtures of sensitive and less-sensitive (resistant) isolates of the pathogens will be co-inoculated on lemon fruit. Once fungal sporulation occurs on the fruit, spores will be harvested, and plated out onto non-amended agar media. Ten to 15 single-spore isolates will then be tested for their fungicide sensitivity. The ratio of numbers of susceptible and resistant single-spore isolates will be used to determine the fitness of resistant isolates in mixtures with sensitive isolates. C. Continue to characterize fungicide resistance mechanisms in Penicillium spp. - Evaluate mutations in the cytochrome P-450 demethylase gene, the target of DMI fungicides, among DMI-resistant. Isolates of P. digitatum will be selected that are resistant against several DMI fungicides (e.g., imazalil, fenbuconazole, propiconazole, CN-2010A) or show differential sensitivities with high resistance against one fungicide, but low resistance against another one. A portion of the cytochrome P-450 demethylase gene where mutations conferring resistance commonly occur will be amplified, sequenced, and mutations will be identified. As an outcome from this research we try to determine if all resistant isolates have the same mutation or if different mutations are involved in resistance development. If mutational differences between different DMI fungicides exist, anti-resistance strategies could be developed by designing pre-/postharvest fungicide rotation programs. III. Develop molecular identification methods for Penicillium spp. on citrus in California and for characterizing populations of G. citri-aurantii. Compare isolates based on RFLP, RAPD, and DNA sequence analyses using isolates sensitive or resistant to selected fungicides. In our research on air sampling there was no problem identifying cultures during the summer months with high population levels of P. digitatum in packinghouses. In the winter months, however, a greater number of contaminating species of Penicillium and other fungi were present in air-samples. Thus, we were successful in developing a semiselective medium that suppresses growth of other fungi for a certain period of time. Still, a molecular assay will allow for rapid and accurate discrimination of pathogen populations from common air-borne populations of Penicillium spp., especially for P. italicum that can be confused with other species that have blue-green spores (e.g., P. commune, P. expansum). This will be important to accurately assess any resistance development in P. italicum. For G. citri-aurantii, a large database on molecular diversity based on AFLP analyses among over 90 isolates has been generated that will be analyzed using procedures of numerical taxonomy. This will allow to make conclusions on the population structure and its variability. Thus, a high level of variability in the population may indicate that the population is either sexually reproducing or it may easily accumulate mutations. This could indicate a high risk for fungicide resistance to develop because a high genetic variability in random DNA sequences may likely reflect a high variability in genes related to fungicide sensitivity. Research Collaboration* (be specific): Collaboration is also being obtained from service companies and registrants of fungicides. Technology Transfer* (be specific): Information is being transferred to industry through scientific publications, oral presentations at meetings with growers, packers, or industry representatives. Other Funding Sources for this Project (current and/or pending)*: None
Project Budget Department Account Number: (if applicable) Salaries and Benefits: Year: 2010-2011 Year: 2011-2012 Year: 2011-2013 Postdocs (To be announced -50%)/ Project Scientist (H. Forster) 20% time Research Assistants (Undergraduate Students) 49% time 30,000 30,000 30,000 3,000 3,000 3,000 SRAs (Dan Felts -10% time) 0 0 0 Lab/Field Assistance 0 0 0 Benefits 12,000 12,000 12,000 Supplies and Expenses: 2,000 2,000 2,000 Equipment: 1,000 1,000 1,000 Operating Expenses/Travel: 3,000 3,000 3,000 Lindcove Recharges: 0 0 0 Lindcove Packline: 0 0 0 Other: None 0 0 0 ANNUAL TOTAL: 51,000 51,000 51,000 Signatures Project Leader: Date: July 31, 2010 Dept. Chair: Date: July 31, 2010 (If applicable)