LOW PRESSURE CONDITIONS KILL QUARANTINE INSECTS WHILE MAINTAINING OPTIMUM TROPICAL FRUIT QUALITY DURING SHIPMENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0211739
• Project Start Date: Sep 1, 2007
• Project End Date: Oct 31, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
TROPICAL RESEARCH & EDUCATION CENTER, HOMESTEAD

Non Technical Summary
Insect pests that endanger our domestic food supply are a vital concern of American agriculture. The U.S. and other countries prohibit importation of horticultural commodities that have not been treated by an approved method ensuring Probit 9 mortality of quarantine pests. Because the widely used methyl bromide fumigation method is being phased out, and other quarantine treatments degrade the quality of imported fruits and vegetables, a new approach is needed. Determining the shortest time required for low pressure (15mm Hg) to kill fruit-borne eggs and larvae of Caribbean fruit flies and other fruit fly species at the probit 9 level security will establish low pressure as a UDSA/APHIS-approved phytosanitary treatment.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	1099	1020	20%
501	3110	1020	30%
503	1099	1020	20%
503	3110	1020	30%

Knowledge Area
501 - New and Improved Food Processing Technologies; 503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
1099 - Tropical/subtropical fruit, general/other; 3110 - Insects;

Field Of Science
1020 - Physiology;

Keywords

hypobaric storage

vacuum intermodal container

vacufresh

fruit fly

insect suffication

insect mortality

tree ripe fruit

vine ripe vegetables

optimum postharvest quality

commodity shipment

mature fruit shipment

mango

papaya

Goals / Objectives
Research objective #1. Determine the time required for low pressure (LP) to kill Caribben fruit flies in tropical fruit. The working hypothesis for this objective is that the optimal low pressure condition for tropical fruit storage will cause Caribbean fruit fly eggs and larvae to die inside guavas, mangoes, and papayas as efficiently as it kills them on agar medium. Research objective #2. Determine the time for LP to kill various fruit fly species that infest tropical grown in tropical America and Hawaii. The working hypothesis for this objective is that eggs and larvae of fruit flies that infest guava, mango, papaya, and other tropical fruits will be killed at Probit 9 level within the time-frame required for a shipment of these fruits to mainland US from tropical America or Hawaii. Research objective #3: Determine the time to kill fruit flies present in tropical fruits, without compromising fruit quality, when the fruits are sequentially vacuum-cooled and then stored in the same LP chamber. The working hypothesis for this objective is that vacuum-cooling in an LP container prior to sequentially storing tropical fruits at their optimal LP condition will shorten the time to kill insect pests at Probit 9 level without compromising the quality of the stored fruit.

Project Methods
Low pressure (LP) air changes at 15 mm Hg will be flowed through stainless steel laboratory LP chambers at one volume per hour and humidified by bubbling the expanded LP air through heated water before it enters the storage chamber. This will keep the relative humidity close to saturation provided that the system is leak-tight. Fittings and seals specially designed for vacuum applications will be used in order to avoid leakage, and mini thermistor sensor probes and a universal readout meter will constantly indicate the dry- and wet-bulb temperatures within each chamber. Insect eggs will be obtained from local insect rearing facility and transferred into 10 ml disposable syringes using established methods. A 0.1 ml aliquot (~100 eggs) will be dispensed into each oviposition site described below. Mature guavas, mangoes, and papayas will be harvested during appropriate production seasons from field plants at the Tropical Research and Education Center, or obtained from local packing houses and by purchase from local grower orchards or grocers, as required depending upon research location. The best inoculation method to ensure that eggs will hatch into viable larvae will be determined. A deposit 0.1 ml of an egg suspension into an 8-mm hole cut near the fruit center with a cork borer or by a surface method wherein a recess under an epidermal flap will be created using a scalpel and the egg suspension deposited. Regardless of the inoculation method, each guava and mango will receive approx. 100 eggs, and the same number will be placed into each of five sites evenly distributed around American type papaya fruits. In each experiment, 30 guavas, mangoes, or Solo papayas, and six American type papaya fruit per exposure time will be inoculated with eggs or larvae and exposed for 2, 4, 6, 8, 10, and 12 days in leak tight stainless steel containers precisely regulated to 15 mm Hg and 13 C with a metered airflow allowing one rarified air exchange/hr. Thirty pre-cooled atmospheric-pressure-treated fruits per exposure time will be enclosed and flushed with one humidified 13 C air-change per hour in the same intermodal container. The fruit from each exposure treatment will be transferred to a plastic bin containing one-half inch of fine vermiculite in its bottom to facilitate pupation of emerging larvae. The bin will incubated in the 25 C intermodal container for two weeks (one week for larvae) before the vermiculite will be sifted to separate out pupae for counting. A non-treated control, common to both sets of treatments, will consist of 30 egg- or larvae-inoculated fruit (six in the case of the American papaya) of each fruit type to be incubated within the 25 C intermodal container. At least 10 experiments will be performed for eggs and larvae inoculated into each type of fruit at each pressure. A probit analysis (PROC GENMOD, SAS for Windows v9.1, 2003, SAS Institute, Inc, Cary, NC or similar statistical packaged program) will be used to analyze the normalized mortality rates vs. time in response to the 13 C LP and AP treatments in order to predict the time required to achieve 99.999% mortality with a confidence level of 95%.

Progress 09/01/07 to 10/31/10

Outputs
OUTPUTS: Davenport, T. L. is no longer with University PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Davenport, T. L. is no longer with University

Publications

• No publications reported this period

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Fresh, horticulturally mature commodities can be stored far longer using low pressure, flow-through technology than any other method of storage. Low-pressure intermodal shipping containers will allow these products to be transported to markets across the world such that they will ripen to fresh-picked flavor and quality regardless of distance traveled or shipment time. Previously published research demonstrated that the conditions optimal for storing fresh tropical fruit at low pressure also killed Caribbean fruit fly (CFF) eggs and larvae on agar insect media by 9.5 days. This conclusion opens the possibility that hitch hiking quarantine insects infesting tropical and temperate fruits, vegetables, and flowers can be completely killed during low-pressure shipments. The primary purpose of the current project was to determine if these optimum shipping conditions of 13C, ~0.13% oxygen flowing through the system at one rarified air exchange per hour at 15 mm Hg, and nearly 100% relative humidity provided during low-pressure storage can also kill insect eggs and larvae in fruit. CFF eggs were placed in mango, papaya, and guava fruit and tested for mortality over time in a variety of optimal to non-optimal fruit storage conditions. The rate of CFF egg mortality at low pressure over time was the same as those at the same temperature at atmospheric pressure (760 mm Hg). Because the fruit produced acetaldehyde and ethyl alcohol due to fermentation at low pressure, static, non-flow-through systems allowing alcohol accumulation in the vacuum chambers were tested with a 98% mortality by 15 days. Saturating the vacuum chambers with ethyl alcohol vapor at 25 mm Hg killed 100% of CFF eggs on water-wetted filter paper by 24 hr exposure; however, condensation of the alcohol vapor in the water over time, thus building alcohol concentrations to about 70%, was necessary for complete mortality. Such concentrations are higher than fruit pulp can tolerate. Other compounds known to be toxic to insects, such as ethyl formate and ethyl acetate are next to be tested for efficacy in killing the CFF eggs. Manuscripts will be prepared to describe the role and control of commodity fermentation and insect mortality in low-pressure storage. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Target audiences are scientists involved in postharvest physiology and quarantine control of invasive insects as well as producers and shippers of horticultural commodities. The research is still evolving, but it is anticipated to deliver innovative quarantine treatments to prevent invasive insects from entering the United States with horticultural imports. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Results have led to a number of unanticipated discoveries regarding commodity storage at low pressures. It was previously assumed that unlike normal atmospheric pressure, the constantly low oxygen levels in a vacuum were able to readily enter the fruit due to the low resistance to diffusion of gases in the rarified atmosphere in order to maintain aerobic respiration. We discovered that the fruit was only partially respiring aerobically in a vacuum, but mostly fermenting depending on the oxygen partial pressure. The constant flushing of the end products of fermentation, i.e. CO2, ethyl alcohol, and acetaldehyde from the system apparently prevents sufficient build up of these products to cause commodity damage. This led to a series of experiments to determine if the alcohol build up could be sufficient to kill insect eggs embedded in and on the surface of fruit. We concluded that it does not. A 98% kill in 15 days is insufficient kill rate to satisfy the probit 9 level of kill (99.97%) required for an acceptable quarantine treatment. In order to determine the lethal time of exposure of eggs to alcohol, we discovered that the vapor itself does not kill the eggs. Instead, the concentration of condensing alcohol builds up in the water over time in the vacuum. The time to kill coincided with about a 70% level of ethanol built up in the Petri dishes holding the filter paper discs wetted with half ml water. The water accumulated more than 1 ml of liquid ethanol over the 24 hr exposure period in accordance with Henry's law. It is still not clear why insects eggs and larvae are readily killed in vacuum on artificial agar media while not being killed in fruit stored at the same low pressures. Research is continuing in an effort to better understand these results, but it is becoming clear that ethanol does not appear to be a promising toxic agent in vacuum systems. The levels inside fruit will have to be too high to kill the insect eggs without also killing the fruit. Other natural products produced by plants that are highly toxic to insects, such as ethyl formate, may prove efficacious in killing the insects while preserving the quality of the stored commodity.

Publications

• Davenport, T. L., Burg, S. P., and Follett, P. 2008. A Lab Scale Low-pressure Chamber System for Conducting Hypobaric Research. HortScience 43:1168. Abstract.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: A USDA/ARS Specific Cooperative agreement was entered between my laboratory at the University of Florida, IFAS, Tropical Research and Education Center and the USDA Quarantine Research Laboratory, Hilo, Hawaii in early 2007. Funding ($30,200) was received in mid November 2007 to construct the containers and complete experiments designed to determine the time to kill Caribbean fruit fly (CFF) eggs and larvae in guava, mango, and papaya fruit. Eight leak-free aluminum vacuum chambers were designed with an internal volume of 2'x2'x1.5', and accompanying humidifiers were designed to provide near 100% humidity to each vacuum chamber regardless of the quantity of stored commodities. The containers, fabricated by Atlas Technologies, Port Townsend WA, arrived in mid August 2008. Final construction of the plumbing linking the vacuum pump, pressure regulator, humidifier heaters, and the associated control and monitoring devices and testing of the equipment has now been completed. The first CFF experiments will begin in mid November 2008. This research follows published results demonstrating a probit 9 kill of CFF eggs and larvae on agar medium within 9.4 days by exposure to 15 mm Hg at 13 degrees C and one rarified air exchange per hour. These same conditions preserve the fresh-harvested quality of virtually all horticultural commodities for months. PARTICIPANTS: Stanley P. Burg, the inventor of hypobaric storage, serves as a non-paid consultant on this project. TARGET AUDIENCES: Target audiences include: Postharvest physiologists Shippers and handlers of fresh produce PROJECT MODIFICATIONS: No major changes in protocols are planned. Progress is underway.

Impacts
One of two prototype commercial VacuFresh intermodal containers providing the same conditions as the lab-scale containers described above is now available. Our overall objective is to qualify shipments of exported and imported commodities in these low-pressure containers as the quarantine treatment of choice for invasive insects of all types. The time to kill CFF in fruit is anticipated to be similar to that on media (9.4 days) if fruit are pre-cooled to 13 degrees C before loading them with eggs or larvae. If eggs or larvae are loaded into the fruit at room temperature before exposing them to low pressure, they should be completely killed by about 5 days, well within the time for the shortest transits to market on container ships. Once the time-to-kill data are fully collected for the CFF in Homestead, four of the vacuum chambers will be shipped to Hilo, Hawaii to continue the research on the four fruit fly pests infesting that state.

Publications

• No publications reported this period