Specialty Crops Research Program

Specialty Crops Research Program

Annual Report:

Report Period: Inception of Project – December 31, 2003

Project Title: Evaluation of food additives and low-toxicity compounds as alternative chemicals to synthetic fungicides for the control of the main postharvest diseases of California stone fruits.

Principal Investigator (s): Dr. Carlos H. Crisosto; University of California, Davis; Kearney Agricultural Center, 9240 S. Riverbend Ave., Parlier, CA 93648; e-mail: carlos@uckac.edu; Tel. (559) 646-6596; Fax. (559) 646-6593

EXECUTIVE SUMMARY.

Stone fruits (peach, nectarine, plum) are major crops in California. Economic losses caused by postharvest diseases are among the most important concerns of the growers. Postharvest fruit decay has typically been controlled by application of synthetic fungicides. However, important problems associated with the massive use of these chemicals, such as the proliferation of resistant strains of the pathogens and concerns about public health and environmental contamination, have increased the need for alternatives, especially in the context of Integrated Pest Management (IPM) practices in California. In this project, we are undertaking the evaluation of a wide range of food additives and low-toxicity chemicals as potential alternatives to synthetic fungicides for the control of the most important postharvest pathogens of stone fruits. These compounds leave low or non-detectable residues on the fruit and are approved for many industrial and agricultural applications by Federal and State regulations. Many of them are registered as Generally Recognized as Safe (GRAS) by the EPA, or are included in the National List of substances allowed as ingredients in products labeled as organic.

Our project has three objectives that involve a sequential screening process. The goal is to set the basis for the commercial implementation of reliable and cost-effective alternative treatment(s) for the control of target postharvest diseases of stone fruit crops in California. In the first year, we have performed an initial screening of 25 chemicals on 7 stone fruit cultivars against 7 pathogens (Botrytis cinerea, Monilinia fructicola, Geotrichum candidum, Penicillium expansum, Alternaria alternata, Rhizopus stolonifer, and Mucor piriformis) in vivo (Objective 1). Eighteen GRAS compounds were eliminated in the screening due to lack of effectiveness in controlling pathogens or damage to fruit. One remaining compound (acetic acid) requires further study before being either eliminated or advanced. Six GRAS compounds (2 deoxy – D – glucose, potassium carbonate, potassium sorbate, sodium carbonate, sodium sorbate, sodium benzoate) will be advanced to Objective 2 for further testing in 2004. Here, they will be evaluated to determine the most effective solution temperature, chemical concentration, and immersion period. Postharvest treatments selected under Objective 2 will be further tested during cold storage of the treated fruit

Crisosto et al., 2003 SCRP Annual Report

(Objective 3). Five of the GRAS compounds that are being advanced have good activity against Botrytis cinerea and two have limited activity against Monilinia fructicola. Several of the 6 GRAS compounds also have activity against Geotrichum candidum. One of the 6 compounds has some control against Rhizopus stolonifer.

OBJECTIVES.

Our project is divided into three main objectives that involve a sequential screening process. The goal is to set the basis for the commercial implementation of a reliable and cost-effective alternative chemical treatment for the control of target postharvest diseases of stone fruit crops in California.

Objective 1 (2002-2003).

Evaluate in in vivo primary screenings the effectiveness of a wide range of low-toxicity chemicals, mostly common food additives, for the control of the main postharvest pathogens of peach, nectarine, and plum.

Objective 2 (2004-2005).

Evaluate in small-scale trials (dips in aqueous solutions) the effectiveness of chemicals selected in Objective 1. Determine the most appropriate combination of solution temperature, chemical concentration, and immersion time needed to provide optimal disease control.

Objective 3 (2004-2005).

Evaluate in small-scale trials (dips in aqueous solutions) the effectiveness of chemicals selected in objective 2 on fruit treated then stored at low temperature. Assess decay periodically during the cold storage period, and decay and fruit quality at the end of the storage period and after a shelf life period. Study the commercial feasibility of these treatments and set the basis for commercial-scale evaluation trials.

PROCEDURES AND METHODS.

Objective 1. In vivo primary screenings.

Fruit. GRAS chemicals were evaluated on cherries (‘Brooks’), peaches (‘Flavorcrest’, ‘O’ Henry’, ‘Last Chance’), nectarines (‘Summer Fire’) and plums (‘Fortune’, ‘Royal Diamond’). Fruit were sanitized with 100 ppm free sodium hypochlorite, rinsed and packed in commercial tray packs/boxes to dry prior to inoculation by fungal pathogens (Table 27, Plate 2) and treatment with GRAS compounds (Plate 3). Fruit were used after drying or stored at 1°C for up to three days prior to inoculation.

Fungal inoculum. Pure cultures of the main postharvest pathogens were maintained on refrigerated, acidified Potato Dextrose Agar (PDA). These pathogens included Botrytis cinerea, Monilinia fructicola, Geotrichum candidum, Penicillium expansum, Alternaria alternata, Rhizopus stolonifer, and Mucor piriformis. Spores were harvested from the

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mycelial growth when plates were from 4 to 10 days old depending upon the growth rate of the fungal species (Table 27). Spores were washed from the plates with sterile water then filtered through two layers of cheesecloth. The aqueous solutions were calibrated using a hemacytometer to a spore density of 5 X 10⁴/ ml, with the exception of G. candidum, which was prepared at 1 X 10⁸/ ml. Spore suspensions were applied to a single, sterile, 5mm deep by 2mm wide wound on the cheek of each fruit at the rate of 10µl for cherries and 20µl for the other fruits. After inoculation, fruit were incubated at room temperature for 15 to 18 hours prior to the application of the GRAS solutions to the wound site.

GRAS compounds. GRAS chemicals (Table 1.) were prepared as 1 M stock aqueous solutions (Table 27). Dilutions were prepared as molar dilutions. Aqueous acid solutions were diluted as percentages of the stock solution. All solutions were filter sterilized with 0.45µm syringe filters. Solutions were dispensed by repeating-pipette for uniformity of application at the rate of 20µl for small fruit or 40µl per large fruit.

Evaluation. Treated fruit were incubated at 20°C and assessed after 3 and 5 days for disease incidence and severity. Fruit were measured for severity with the diameter of the fungal infection on each fruit measured with an electronic caliper. Treatment means were compared with SAS using General Linear Models (GLM) regression.

MAJOR ACTIVITIES.

Year 1

? February-May 2003: Purchase of chemicals and laboratory preparation for experiments in Year 1.

? May-June 2003: Preliminary screening of GRAS chemicals conducted using ‘Brooks’ cherries.

? June-September 2003: Objective 1 screening with ‘Flavorcrest’, ‘O’ Henry’ and ‘Last Chance’ peaches; ‘Summer Fire’ nectarines; and ‘Fortune’ and ‘Royal Diamond’ plums.

? September-December 2003: Data analysis, progress report, purchase of chemicals and laboratory preparation for experiments in Year 2.

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SIGNIFICANT OBSERVATIONS.

Of the 25 compounds tested on stone fruit, 18 have been eliminated and 6 (2 deoxy – D – glucose, potassium carbonate, potassium sorbate, sodium carbonate, sodium sorbate, sodium benzoate) will be tested in Objective 2. Acetic acid will require further study before being either eliminated or advanced. All six of the GRAS compounds that are being advanced have good activity against Botrytis cinerea, and all have limited activity against Monilinia fructicola, with the exception of sodium carbonate, which has good activity. Geotrichum candidum is also controlled by 6 GRAS compounds. Deoxy D glucose has some control against Rhizopus stolonifer, and acetic acid vapor reduces the growth of R. stolonifer. Current focus is to complete evaluations after incubation and analyze data to clearly identify compounds and concentrations that control the postharvest pathogens on each of the fruit species.

PROBLEMS.

Research was complicated this year by a high incidence of natural, quiescent infections. However, all research objectives were met.

BUDGET SUMMARY.

Our expenses charged to this project to date (excluding overhead) are as follows:

Expense category	Budgeted	Actual
Personnel	$ 42,039	$ 36,252
Benefits	$ 10,509	$ 13,187
Supplies and Expenses	$ 6,560	$ 3,348
Equipment	$ 1,280	$ 1,100
Travel	$ 1,000	$ 0
TOTAL	$ 61,388	$ 53,887

Our estimated budget activity for the next quarter includes $15,000 in personnel salary and benefits.

DOCUMENTATION OF ACTIVITIES.

A summary of GRAS compound efficacy and phytotoxicity is presented in Tables 1-26 in the appendix. A flow chart of the experimental protocol is presented in Table 27 in the appendix. A visual representation of the inoculation and evaluation procedure is presented in Plates 1-9 in the appendix.

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APPENDIX.

Table 1. GRAS chemicals and concentrations evaluated in primary screening trials, 2002-2003.

Chemical	Concentrations evaluated	Advanced to next stage
Acetic acid (vapor)	300mM @200µL= 3.6X10^-3g/15L	Possible. Good activity against pathogens, delivery system needs work
Ammonium bicarbonate	100, 200, 400 mM	No. No control at any concentration
Ammonium carbonate	100, 200, 400 mM	No. Slight control of a few pathogens
Ammonium molybdate	8, 16, 100 mM	No. Some control, damages and stains
Ascorbic acid	100 mM	No. Little control of most pathogens
Deoxy-D-glucose	25, 50, 100 mM	Yes. Good control of most pathogens
Deoxy-D-ribose	25, 50, 100 mM	No. No control except of Geotrichum
Hydrogen peroxide	30, 140, 340 mM	No. Damage to tissue at all conc.
Lactic acid	8 mM	No. Damage to tissue, increases infection
Potassium acetate	30, 100, 300 mM	No. No control of pathogens
Potassium benzoate	20, 100, 200 mM	No. Some control of Botrytis at high conc
Potassium bicarbonate	100, 200, 400 mM	No. No control of pathogens
Potassium carbonate	100, 200, 250 mM	Yes. Some control of Botrytis, Monilinia
Potassium propionate	20, 100, 200 mM	No. No control of pathogens
Potassium sorbate	20, 100, 200 mM	Yes. Some control of Botrytis, Monilinia
Sodium acetate	100 mM	No. No control except Geotrichum
Sodium benzoate	20, 100, 200 mM	Yes. Some control of pathogens
Sodium bicarbonate	100, 200, 400 mM	No. No control of pathogens
Sodium carbonate	100, 200, 400 mM	Yes. Some control of Botrytis, Monilinia
Sodium citrate	100 mM	No. No control of pathogens
Sodium lactate	100 mM	No. Damage to tissue, increases infection
Sodium molybdate	12.5, 50, 100 mM	No. Some control, damages and stains
Sodium propionate	30, 100, 300 mM	No. Some control of Botrytis at high conc.
Sodium sorbate	20, 100, 200 mM	Yes. Some control of pathogens
Sodium tartrate	100 mM	No. No control of pathogens

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Plates 1-9. Inoculation, treatment, and evaluation of GRAS chemical compounds. 1. Fruit were wounded to uniform size and depth prior to inoculation with pathogens. 2. A drop of inoculum was placed on the wound site, then infection allowed to become established during an 18 h incubation at 20° C. 3. Eighteen hours after inoculation, chemical treatments were applied. Fruit were then incubated up to 5 d at 20° C. 4. Decay lesion size was measured, recorded and analyzed statistically. 5. Control (untreated) peaches inoculated with M. fructicola then incubated for 5 d at 20ºC. 6. Peaches inoculated with M. fructicola, treated with acetic acid vapor, then incubated for 5 d at 20ºC. 7. Control (untreated) peaches inoculated with B. cinerea, then incubated for 5 d at 20ºC. 8. Peaches inoculated with B. cinerea, treated with sodium benzoate, then incubated for 5 d at 20ºC. 9. Peaches inoculated with B. cinerea, treated with deoxy -D - glucose, then incubated for 5 d at 20ºC. 10