Progress 06/11/02 to 09/01/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Fresh fruit are attacked by fungal diseases that rot fruit after harvest. The fungal diseases that rot fruit after harvest cause losses of about 5% of California's $3 billion production. The control of the disease-causing fungi is important, and is usually accomplished using chemical fungicides and sanitizers, but issues of pest resistance to these chemicals, the dietary safety of their presence in foods, and their impact on the environment has made the search for safer replacements important. Fungicides used to control these diseases interfere with the export of citrus fruit because some countries will not accept residues of the fungicides on the fruit. Three fungicides are approved for citrus fruit use in California, sodium ortho-phenyl phenate, imazalil, and thiabendazole. Sodium ortho-phenyl
phenate is classified as a probable human carcinogen and imazalil as a possible human carcinogen by the U.S. Environmental Protection Agency. We propose to use 'reduced-risk' compounds, such as lime-sulfur solution, sodium bicarbonate, or ethanol, to control these fungi that will minimize the dietary risk posed by fungicides present in fruit purchased by consumers. This work will not only benefit consumers, but produce growers and distributors will also benefit because chemical residues on products are prohibited by some importing countries and in domestic markets where organic produce is sold. 2. List the milestones (indicators of progress) from your Project Plan. Milestones and Expected Outcomes (Objective 1 - Joseph Smilanick) Year 1 (FY2004). Conduct laboratory trials to control postharvest green mold and sour rot on citrus fruit, and postharvest disease control trials with table grapes to control gray mold. Year 2 (FY2005). Conduct large-scale laboratory trials to control
postharvest green mold and sour rot on citrus fruit, and postharvest disease control trials with table grapes to control gray mold. Include rigorous fruit quality evaluation of effective approaches, conduct grower/processor demonstration tests, seek industry input and collaboration, inquire about registration issues with regulatory agencies, and establish the category of regulation applied to the process. Year 3 (FY2006). Conduct pilot-scale trials with naturally infected fruit in collaborator's facilities. Refine treatment protocols to maximize their effectiveness and practicality. Address patent issues. Participate in compliance of registration needs with regulatory agencies and sponsoring registrant. Year 4 (FY2007). Conduct pilot-scale trials with naturally infected fruit in collaborator's facilities with several important cultivars. Refine treatment protocols to maximize their effectiveness and practicality. Participate in compliance of registration needs with regulatory agencies
and sponsoring registrant. Year 5 (FY2008). Continue pilot-scale trials if necessary. Refine treatment protocols to maximize their effectiveness and practicality. Prepare manuscript describing results. Milestones and Expected Outcomes (Objective 2 - Joseph Smilanick) Year 1 (FY2004). Develop of inoculation and culture methods for large- scale powdery mildew inoculation and disease quantification. Conduct anatomical studies of berries surfaces and skin of selections with gray mold resistance. Year 2 (FY2005). Begin large-scale powdery mildew inoculation and disease quantification, establish in which organs resistance is expressed (leaves, rachis, or fruit) and when. Complete anatomical analyses of berries surfaces and skin of selections with gray mold resistance. Year 3 (FY2006). Continue large-scale powdery mildew inoculation and disease quantification trials as in Year 2. Begin analysis of selections where resistance and susceptibility have been established to determine what features
are associated with resistance. Prepare a manuscript describing anatomical features of berries surfaces and skin of selections with gray mold resistance. Year 4 (FY2007). Continue and confirm results from prior seasons of large-scale powdery mildew inoculation and disease quantification trials begun in Year 2. Continue analysis of selections where resistance and susceptibility have been established to determine what features are associated with resistance. Year 5 (FY2008). Conclude analysis of selections where resistance and susceptibility have been established to determine what features are associated with resistance, and prepare a manuscript on this subject. Milestones and Expected Outcomes (Objective 3 - David Obenland and Louis Aung) Year 1- 2 (FY2004 and 2005): Initiate planned heat treatments of stone fruit and lemons; measure quality and phytotoxicity effects; extract collected samples for soluble sugars. Develop database relating fruit color, soluble solids/titratable
acidity, ethanol and navel orange fruit acceptability. Assay antioxidant response to heat treatment and relate to subsequent injury. Year 3-4 (FY2006 and 2007): Purify samples and determine sugars by HPLC; prepare report. Include additional variables such as waxing and handling into navel orange fruit acceptability study. Continue antioxidant assays and identify compounds present in lemon peel that are responsible for antioxidant response. Year 5 (FY2008): Analyze and summarize data; prepare manuscripts for review and publication. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. FY 2004 Milestones. 1. Conduct laboratory trials to control postharvest green mold and sour rot on citrus fruit, and postharvest disease control trials with table grapes to control gray mold.
Progress: Substantially met this milestone: Seven small-scale laboratory tests with citrus fruit, two with the objective to control both green mold and sour rot, were conducted during this period in research to develop alternatives to synthetic fungicides. Four tests to control postharvest decay of table grapes were conducted in the reporting period, using approaches that minimize or eliminate sulfur dioxide use on table grapes. 2. Develop inoculation and culture methods for large-scale powdery mildew inoculation and disease quantification. Conduct anatomical studies of berries surfaces and skin of selections with gray mold resistance. Progress: Substantially met this milestone: An inoculation tower was fabricated to apply powdery mildew inoculum onto grapevine fruit and leaves. The mildew resistance of seventy plant geneticist selections was quantified and compared to the mildew resistance observed for many of these selections in vineyard trials. In general, vineyard resistance to
powdery mildew could be predicted by the resistance of young vines. The behavior of Uncinular necator conidia on the surface of leaves of a susceptible and resistant grapevine leaves was observed by scanning electron microscopy. Conidial germ tube growth stopped briefly after germination on the resistant line, while it proceeded into the leaf in the susceptible line. 3. Initiate planned heat treatments of stone fruit and lemons; measure quality and phytotoxicity effects; extract collected samples for soluble sugars. Develop database relating fruit color, soluble solids/titratable acidity, ethanol and navel orange fruit acceptability. Assay antioxidant response to heat treatment and relate to subsequent injury. B. List the milestones that you expect to address over the next three years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2005. Conduct large-scale laboratory trials to control postharvest green mold
and sour rot on citrus fruit, and postharvest disease control trials with table grapes to control gray mold. Include rigorous fruit quality evaluation of effective approaches, conduct grower/processor demonstration tests, seek industry input and collaboration, inquire about registration issues with regulatory agencies, and establish the category of regulation applied to the process. Begin large-scale powdery mildew inoculation and disease quantification, establish in which organs resistance is expressed (leaves, rachis, or fruit) and when. Complete anatomical studies of berries surfaces and skin of selections with gray mold resistance. Initiate planned heat treatments of stone fruit and lemons; measure quality and phytotoxicity effects; extract collected samples for soluble sugars. Develop database relating fruit color, soluble solids/titratable acidity, ethanol and navel orange fruit acceptability. Assay antioxidant response to heat treatment and relate to subsequent injury.
Anticipated accomplishments: Evaluate several techniques for use in citrus fruit packinghouses and groves to minimize decay losses while reducing or eliminating synthetic fungicide inputs. Identify and quantify powdery mildew resistance in grape vine selections, with microscopic observations describing the appearance and behavior of the powdery mildew pathogen on resistant and susceptible selections. FY 2006. Conduct pilot-scale trials with naturally infected fruit in collaborator's facilities. Refine treatment protocols to maximize their effectiveness and practicality. Address patent issues. Participate in compliance of registration needs with regulatory agencies and sponsoring registrant. Continue large-scale powdery mildew inoculation and disease quantification trials as in Year 2. Begin analysis of selections where resistance and susceptibility have been established to determine what features are associated with resistance. Prepare a manuscript regarding anatomical features of
berries surfaces and skin of selections with gray mold resistance. Purify samples and determine sugars by HPLC; prepare report. Include additional variables such as waxing and handling into navel orange fruit acceptability study. Continue antioxidant assays and identify compounds present in lemon peel that are responsible for antioxidant response. Anticipated accomplishments: Continue evaluation of several techniques for use in citrus fruit packinghouses and groves to minimize decay losses while reducing or eliminating synthetic fungicide inputs. Identify and quantify powdery mildew resistance in grape vine selections, with microscopic observations describing the appearance and behavior of the powdery mildew pathogen on resistant and susceptible selections. FY 2007. Conduct pilot-scale trials with naturally infected fruit in collaborator's facilities with several important cultivars. Refine treatment protocols to maximize their effectiveness and practicality. Participate in
compliance of registration needs with regulatory agencies and sponsoring registrant. Continue and confirm results from prior seasons of large-scale powdery mildew inoculation and disease quantification trials begun earlier. Continue analysis of selections where resistance and susceptibility have been established to determine what features are associated with resistance. Anticipated accomplishments: Participate with citrus industry partners in the introduction of several techniques for use in citrus fruit packinghouses and groves to minimize decay losses while reducing or eliminating synthetic fungicide inputs. Continue to identify and quantify powdery mildew resistance in grape vine selections, with microscopic observations describing the appearance and behavior of the powdery mildew pathogen on resistant and susceptible selections. 4. What were the most significant accomplishments this past year? A. The California citrus industry needs to market the highest quality fruit in order
to stay competitive in the marketplace. In a collaboration between David Obenland (ARS-Parlier) and Mary Lu Arpaia (University of California, Kearney) various quality parameters were evaluated during fruit maturation and following different industry postharvest treatments and comparisons made to the sensory acceptability of the fruit as determined by taste panels. Acceptability of the fruit was shown to increase during maturation and to be closely related to the level of sweetness and acidity. This information may be used to support efforts to change the minimum maturity requirements that govern when oranges may be harvested in California. B. Freeze injury caused economic losses to itrus growers and packers and the industry needs a simple and inexpensive test for determining freeze injury on quality of oranges. Scientists of the Postharvest Quality and Genetics Research Unit in collaboration with support of the Citrus Research Board conducted tests of freezing temperatures on
volatile emissions from navel oranges. Ethanol and three other chemical compounds were found in frozen oranges while none were detected from unfrozen fruits. The findings could provide the citrus industry a rapid method to differentiate marketable quality fruit from injury fruit. Ozone gas is used commercially to prolong the storage life of fresh fruit, but its ability to penetrate into packages was unknown. The penetration of ozone gas into commercial citrus fruit packages was determined with commercial collaborators and Dr. Lluis Palou of the University of California, Davis. Ozone was measured by a multiple-channel ozone analyzer within many packages over a period of weeks, and several of the best packages to use with ozone were identified. This work provides key information to use ozone gas intelligently in fruit storage facilities. Postharvest decay of avocado fruit destroys a significant portion of this crop. The fungi that cause these losses have not been systematically
identified and surveyed. In collaboration with Dr. Mary Lu Arpaia of the University of California, Riverside, we found most important fungi that rot California avocados after harvest were Dothiorella, Colletotrichum, and Alternaria. This work identified which pathogens are present is fundamental to develop cultural or chemical methods to minimize postharvest decay losses of avocado fruit. Gray mold, caused by Botrytis cinerea, causes serious pre- and postharvest losses every year to grapes worldwide. Scientists of the Postharvest Quality and Genetics Unit examined the natural resistance of grape berries of many table grape selections to infection, then measured many chemical and anatomical attributes of berries associated with resistance. We found few or no pores on the berry surface and the thickness of the skin were consistently associated with resistance to gray mold. This finding facilitates the selection of grape cultivars that resist this pathogen, which is a preferable means
to manage this disease rather than the chemical approaches now in use. Powdery mildew of table grapes causes significant losses in grape yield and quality every year, and the fungicide applications to vines to control this disease are very many. Scientists of the Postharvest Quality and Genetics Unit examined the use of natural mildew resistance. We developed a method where the resistance of young vines, determined with 2-year old vines, generally predicted that of older vines of four or more years in age. This finding will accelerate the development of resistant vines should reduce the fungicide applications needed. Sanitation of most fresh produce is done with chlorine, which imparts off-flavors, its vapors irritate workers, and chemical disinfection by- products of chlorine are of human health concern. Scientists of the Postharvest Quality and Genetics Unit evaluated ethanol as a conceivable chlorine replacement. We established the toxicity of brief exposures of spores of four of
the most common pathogens of produce (Rhizopus stolonifer, Aspergillus niger, Botrytis cinerea and Alternaria alternata) to heated, aqueous ethanol solutions was quantified and modeled. This work shows that ethanol, a simple natural compound, can be used as an effective agent to control these pathogens. Postharvest decay of citrus fruit by fungal pathogens is a serious problem typically managed by synthetic fungicides. In collaboration with Dr. Julien Mercier of AgriQuest Inc. in Davis, California we examined a new biological control approach termed 'biofumigation'. Postharvest decay of citrus was reduced more than 90% by exposure of lemons to natural fungal volatiles from cultures of the fungus Muscodor albus applied after harvest during storage of the fruit. This work indicates this may be a promising approach for citrus growers to manage postharvest decay losses. C. Significant Accomplishments/Activities that Support Special Target Populations: None D. Progress Report
opportunity to submit additional programmatic information to your Area Office and NPS (optional for all-in-house ('D') projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Our major accomplishments, with credit shared with collaborators, are the continued discovery, development, and transfer of technologies to replace or reduce synthetic fungicide use by the citrus industry, and the identification of disease resistance in new grape selections. These accomplishments are in concordance with the objectives 1, 2 and 3 of Project 5302-43000-029-00D (old number) 'Emerging Technologies to Maintain Postharvest Quality and Control Decay of Fresh Commodities', and with the Action Plans of National Programs 303 and 306. Use of heated imazalil aqueous solutions of the fungicide, that enable its rates to be reduced by 50% or more, is in
common commercial use now as a result of this project. Immersion of fruit in bicarbonate and carbonate solutions, which avoids fungicide use entirely, or in combination with low rates of the fungicides, has also become popular. Promising technologies that are yet to be implemented include 'biofumigation' with Muscador albus and integrated treatments incorporating biological control and thermal curing. In addition to reducing fungicide use, these techniques have improved management of postharvest citrus and table grape diseases, particularly for high value export fruit during long transit. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Beginning in February 2004, a new technology that entailed the application of a high volume, low pressure overhead
drench solution (8 million carton per year output, about 10% of California'a citrus production) was developed. Technology is available to citrus packing- houses in California and elsewhere. This treatment will reduce postharvest decay of the fruit by Penicillium and other pathogens. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. 1. ' Relationship between SSC/TA ratio and acceptability of navel orange.' Presented to the University of California, Plant Sciences Department, Pest Control Management Seminar at Santa Barbara. May 18, 2004. 2. 'Brief heated imazalil treatment of fresh citrus fruit' was presented to a packinghouse manager meeting organized by Dave Sorensen and others of Fruit Growers Supply, Visalia, California. October 22, 2003. 3. 'Fungi isolated from decayed California avocados' presented at 'Avocado Brainstorming', an international meeting organized by Mary Lu Arpaia of Plant
Sciences Department of University of California, Riverside, and others. Ventura Marriott Hotel, Ventura CA October 31, 2003. 4. 'Use of ozone in fruit packinghouses' presented at 2003 Postharvest Conference presented by Washington State University and the Washington State Horticulture Association. Wenatchee Convention Center, Wenatchee WA, December 3, 2003. 5. 'Commercial implementation of biological control in the California citrus packinghouses to control postharvest decay.' Presentation to the Congress 'Development of Biological Control Agents of Diseases for Commercial Applications in Food Production Systems' in Seville, Spain, March 25, 2004. 6. 'Preharvest measures to control postharvest decay of citrus fruit in California.' presented in the Department of Agricultural Sciences, Uludag University, Bursa, Turkey, April 2, 2004. 7. 'Potential commercial applications of 'biofumigation' in postharvest horticulture' Presentation and discussion with Agriquest Co. personnel,
Davis CA December 5, 2003. 8. 'Recent postharvest decay control experiments in California.' Presented to the University of California, Plant Sciences Department, Pest Control Management Seminar at Santa Barbara, May 17, 2004.
Impacts
(N/A)
Publications
- Karabulut, A.K., J.L. Smilanick, F. Mlikota-Gabler, M. Mansour, S. Droby. 2003. Near-harvest applications of Metschnikowia fructicola, ethanol, and sodium bicarbonate to control postharvest diseases of grape in central California. Plant Disease, 87(11):1384-1389.
- Plaza, P., Usall, J., Smilanick, J.L., Lamarca, N., Vinas, I. 2004. Combining pantoea agglomerans (cpa-2) and curing treatments to control established infections on penicillium digitatum on lemons. Journal of Food Protection 67:781-786.
- Mlikota-Gabler, F., Smilanick, J.L., Mansour, M., Mackey, B.E. 2004. Survival of spores of rhizopus stolonifier, aspergillus niger, botrytis cinerea, and alternaria alternata after exposure to ethanol solutions at various temperatures.. Journal of Applied Microbiology 96:1354-1360.
- Mlikota-Gabler, F., Smilanick, J.L., Mansour, M., Ramming, D.W., Mackey, B. E. 2004. Correlations of morphological, anatomical, and chemical features of grape berries with resistance to botrytis cinerea. phytopathology. Phytopathology 93:1263-1273.
- Palou, L., Smilanick, J.L., Crisosto, C., Mansour, M., Plaza, P. 2004. Ozone gas penetration and control of the sporulation of penicillium digitatum and penicillium italicum within commercial packages of oranges during cold storage. Crop Protection 22:1131-1134.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Fresh fruit are attacked by fungal diseases that rot fruit after harvest. The control of the disease-causing fungi is important, and is usually accomplished using chemical fungicides and sanitizers, but issues of pest resistance or immunity to the chemicals, the dietary safety of these chemicals on foods, and their impact on the environment has made the search for safer replacements important. We propose to use 'reduced-risk' compounds, such as lime-sulfur solution, sodium bicarbonate, or ethanol, to control these fungi. 2. How serious is the problem? Why does it matter? The fungal diseases that rot fruit after harvest cause losses of about 5% of California's $3 billion production. Fungicides used to control these diseases interfere with the export of citrus fruit because some countries will not accept residues of the fungicides on the fruit. Three fungicides are approved for citrus fruit
use in California, sodium ortho-phenyl phenate, imazalil, and thiabendazole. Sodium ortho-phenyl phenate is classified as a probable human carcinogen and imazalil as a possible human carcinogen by the U.S. Environmental Protection Agency. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The objectives listed in the components of two National Program Programs are relevant to this work. We seek new methods to control plant pathogens, a component of Plant Diseases 303 (30%). This work reduces losses and improves quality of products after harvest, a component of New Uses, Quality, and Marketability of Plant Products 306 (70%). 4. What were the most significant accomplishments this past year? A. Most Significant Accomplishment: Imazalil is an approved synthetic fungicide commonly applied to citrus fruit but methods to reduce the amount of the fungicide are needed. Altering the solution pH and temperature were done by
scientists at the Postharvest Quality and Genetics Research Unit at Parlier, California. Results showed optimization of the pH of solutions of the fungicide at 23-25C reduced the amount needed to control decay fungi by 90% or more. The outcome of this work is the reduction of fungicide costs, decay losses by packers, and imazalil residues in citrus products purchased by consumers. B. Other Significant Accomplishments: Freeze injury caused ecomonic losses to citrus growers and packers and the industry needs a simple and inexpensive test for determining freeze injury on quality of oranges. Scientists of the Postharvest Quality and Genetics Research Unit in collaboration with support of the Citrus Research Board conducted tests of freezing temperatures on volatile emissions from navel oranges. Ethanol and three other chemical compounds were found in frozen oranges while none were detected from unfrozen fruits. The findings could provide the citrus industry a rapid method to
differentiate marketable quality fruit from injured fruit. C. Significant Accomplishments/Activities that Support Special Target Populations: None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Our major accomplishment, with credit shared with many collaborators, is the continued introduction of several technologies to replace or reduce synthetic fungicide use by the citrus industry. Use of heated imazalil aqueous solutions of the fungicide, that enable rates of the fungicide to be reduced by 50% or more, is in common use now in many citrus packing operations as a result of this project. Immersion of fruit in bicarbonate and carbonate treatments, which avoids fungicide use entirely, or in combination with low rates of the fungicide thiabendazole, has also become popular. In addition to reducing fungicide use, these techniques and others have improved management of postharvest citrus diseases, particularly for fruit
during long export transit. Currently, other technologies to reduce or replace fungicide use by this industry are in progress. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2004: Continue developing reduced risk postharvest treatments to reduce citrus postharvest losses, and determine the mode of action of preharvest applied inducer compounds to control postharvest decay. FY2005: Conduct and complete large-scale packinghouse tests with high pH activation of 'reduced risk' fungicide pyrimethanil for citrus use. Participate in registration and recommended application methods for the introduction of pyrimethanil into the citrus industry. FY2006: Facilitate the implementation of 'reduced risk' and 'USDA-certified organic' approaches to reduce decay losses among table grapes and fresh citrus fruit in collaboration with growers, their associations, and service providers. 7. What science and/or technologies have been transferred and to whom? When is the
science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The implementation of 'reduced risk' and 'USDA-certified organic' approaches that are the subject of this research that do not require formal EPA registration, such as the use of bicarbonate solutions alone or in combination with fungicides, or the use of generally recognized as safe buffers to optimize the pH of already approved fungicides, is rapid. Other technologies that require formal regulatory approval, such as the postharvest use of ethanol on table grapes or the approval of the new reduced risk postharvest fungicide pyrimethanil for citrus, will require a commercial advocate to be a petitioner for registration and this may take several years. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work.
(NOTE: This does not replace your peer-reviewed publications listed below). Presentation: 'Postharvest Ozone Gas and Aqueous Applications to Sanitize and Extend the Shelf life of Fresh Fruit' Symposium 'Ozone III. Agricultural and Food Processing Applications of Ozone as an Antimicrobial Agent' Symposium sponsored by the International Ozone Association, California State University Agricultural Technology Institute, and others. Audience of about 135. October 30, 2002. Presentation: 'Combinations of old and new technologies to manage postharvest decay of citrus fruit' Part of Workshop 'Linking Farmers to Markets' at the 8th International Congress of Plant Pathology, Christchurch, New Zealand. Audience of about 135. February 6, 2003. Presentation: 'Mortality of microorganisms and insects in ozone' Presentation to HortResearch Laboratory, Mt. Albert facility, Auckland, New Zealand. Audience of about 35. February 7, 2003. Presentation: 'Characterization of Botrytis resistance in table
grapes' Presentation in San Joaquin Valley Table Grape Seminar, Visalia, California, sponsored by the University of California and California Table Grape Commission. February 26, 2003. Presentation: 'Preharvest measures to minimize postharvest decay of citrus fruit' Presented to the University of California, Plant Sciences Department, Pest Control Management Seminar at Santa Barbara. Audience of about 85. May 6, 2003.
Impacts
(N/A)
Publications
- Smilanick, J.L., Aieyabei, J., Mlikota-Gabler, F., Doctor, J., Sorenson, D. Quantification of the toxicity of aqueous chlorine to spores of Penicillium digitatum and Geotrichum citri-aurantii. Plant Disease. 2002. v. 86. p. 509-514.
- Smilanick, J.L., Margosan, D.M., Mlikota-Gabler, F. Impact of ozonated water on the quality and shelf-life of fresh citrus fruit, stone fruit, and table grapes. Ozone Science and Engineering. 2002. v. 24. p. 343-356.
- Smilanick, J.L., Sorenson, D., Mansour, M., Aieyabei, J., Plaza, P. Impact of a brief postharvest hot water drench treatment on decay, fruit appearance, and microbe populations of California lemons and oranges. HortTechnology. 2003. v. 13. p. 333-338.
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