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Submission form for phytosanitary treatments submission number: 2009-tppt-107


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International Plant Protection Convention 2010_TPPT_Jul_60

Thermal conditioning in Bactrocera tryoni eggs (Diptera:Tephritidae)

following hot water immersion Agenda: 18.1.7

SUBMISSION FORM FOR PHYTOSANITARY TREATMENTS
Submission number: 2009-TPPT-107 (Secretariat use only)
Complete the following form, preferably in electronic format, and submit by e-mail to the IPPC Secretariat (ippc@fao.org) no later than 15 October 2009. Please use one form per phytosanitary treatment. An electronic version of this form is available at: https://www.ippc.int/id/137399?language=en. Incomplete submissions will be returned. Please save the completed submission form with the following file name: COUNTRY or RPPO NAME –Title of treatment.doc, prior to submitting to the IPPC Secretariat via e-mail.
(Text in brackets given for explanatory purposes)

Name of treatment

Thermal conditioning in Bactrocera tryoni eggs (Diptera:Tephritidae) following hot water immersion



Submitted by: (Name of national or regional plant protection organization) PPPO

Contact: (Contact information of an individual able to clarify issues relating to this submission, including sources of efficacy data)

Name: Mr. Nacanieli Waqa

Position and organization: Biosecurity Technician

Mailing address: Secretariat of the Pacific Community, Private Mail Bag, Suva, Fiji Islands

Phone: +679 337 0733, Fax: +679 337 0021

E-mail: Nacanieliw@spc.int





Treatment description

Active ingredient

(Brand names alone will not be accepted)

Treatment type

Hot water

Target pest

Bactrocera tryoni (Queensland fruit fly)

Target regulated articles

Eggplant, capsicum, mango, breadfruit, papaya

Treatment schedule

Bactrocera tryoni (Froggatt) eggs were immersed in hot water to determine egg mortality. Eggs were either immersed in water at a constant temperature, or experienced changing temperature at a specified rate of increase and from a specified start temperature. Comparison of the estimated lethal time for 99% kill (LT99) of eggs experiencing different treatments allowed thermal conditioning to be identified and quantified. Conditioning depended on the temperature and duration of treatment, being a maximum near 38°C. Most conditioning appeared to occur during the early part of the exposure to a given temperature. Exposure to lethal temperatures (≥42°C) as a target temperature of 46 or 48°C was approached, contributed significantly to the mortality if the rate of heating was relatively slow. Calculations of egg survival in a mathematical model of the conditioning and lethal thermal responses, correlated well with experimental values in terms of both trends and magnitudes of LT99 values. The thermal conditions prior to disinfestation treatment influence the response to subsequent heat treatment and thus have implications for the specification of postharvest quarantine treatments which are often expressed in terms of a fruit centre target temperature. This does not take into account of the influence of temperatures and exposure times in the range 32–42°C which can have a very significant effect on the time required to reach high levels of mortality

Other relevant information

(This should include any assumptions or extrapolations and the supporting evidence for these)

References

Waddell B. C., V. M. Jones, R. J. Petry, F. Sales, D. Paulaud, J. H. Maindonald and W. G. Laidlaw, 2000. Thermal conditioning in Bactrocera tryoni eggs (Diptera: Tephritidae) following hot-water immersion. Post-Harvest Biology and Technology 21: 113-128.

The following form must be completed in accordance with ISPM No. 28 (Phytosanitary treatments for regulated pests), available at: https://www.ippc.int/id/13399?language=en. Copies of all relevant supporting information and publications should be supplied with the treatment submission, preferably in PDF format for ease of subsequent distribution.
The following refers to the relevant sections of ISPM No. 28 and are numbered accordingly.

3.2 Efficacy data in support of the submission of a phytosanitary treatment

The source of all efficacy data (published or unpublished) should be provided in the submission. Supporting data should be presented clearly and systematically.

3.2.1 Efficacy data under laboratory/controlled conditions (Treatments may be considered without efficacy data under laboratory/controlled conditions if sufficient efficacy data is available from the operational application of the treatment (section 3.2.2) and if no data under laboratory/controlled conditions exists this section may be left blank.)

Pest information

Identity of the pest to the appropriate level, life stage, and if a laboratory or field strain was used

Bactrocera tryoni

Conditions under which the pests are cultured, reared or grown

Adult B. tryoni were field collected and subsequently maintained in the laboratory on yeast, fruit fly bacteria (Enterobacter cloacae Jordon), water and sugar, under natural light at 26±1°C. Fruit fly colony rearing methods were very similar to those described by Clare (1997). Eggs were collected from adult females which oviposited through holes punched in the side of plastic cylinders (30 mm internal diameter by 46 mm long by 1 mm wide). The inside surface of the cylinders were smeared with diet consisting of banana fruit pulp, and were then exposed to the adults for 2 h. Eggs were washed from the cylinders with water, placed on dampened filter paper in a closed petri dish and incubated for 24 h in a photoperiod of 12:12 L:D at 26±1°C for treatment as mature eggs (26–28 h old).

Biological traits of the pest relevant to the treatment



Method of natural or artificial infestation



Determination of most resistant species/life stage (in the regulated article where appropriate)

A kinetic model was developed which uses the experimental data obtained from the constant temperature treatment results. The model is developed in terms of

(1) the extra time that conditioning in the temperature range 30–42°C increases the estimated lethal time required to achieve 99% mortality (LT99) at a target temperature of 46°C

(2) the decrease in LT99 due to lethality in the range 42–46°C which results in insect mortality as measured at 46°C. The mortality of fruit fly eggs associated with any temperature ramp from 30 to 46°C can be described by this model.


Regulated article information

Type of regulated article and intended use

Eggplant, mango, capsicum, breadfruit and papaya – potential export commodities.

Botanical name for plant or plant product (where applicable)

Solanum melongena (eggplant)

Mangifera indica (mango)

Capsicum annuum (capsicum)

Artocarpus altilis (breadfruit)



Carica papaya (papaya)

Conditions of the plant or plant product



Experimental parameters

Level of confidence of laboratory tests provided by the method of statistical analysis and the data supporting that calculation

Statistical methods for assessing LT99:

Analyses of observed mortality due to treatment at individual temperatures from 42 to 48°C used the model: log(_log(1_p))_a_bt, (1) where p was the expected observed mortality, and t was the treatment time in minutes (Preisler and Robertson, 1989). This gave approximate linearity in time. The estimated LT99, which relates to mortality after there has been allowance for control mortality, was calculated to give an expected mortality of c_(1_c)0.99, where c was the control mortality as determined in the 25°C immersion treatments. Eq. (1) was fitted using a robust version of the generalized linear model analysis available in S-Plus (Chambers and Hastie, 1991) that assumed that any variance was proportional to that of a binomial distribution. The robust version reduces the weight given to points lying away from the main body of the data. This method handles the variability in the response associated with occasional large outliers (Maindonald, 1992). Confidence intervals were calculated so that non-overlap was equivalent to a statistically significant difference in a Student’s t-test at the 1% level. Although the log–log analysis of Eq. (1) can be used successfully to determine LT99 times we have utilized a kinetic expression for mortality to model the response of B. tryoni eggs over the entire treatment history. Rather than use percentage mortality we shall use its complement, percentage survivors, S(T, t)_100 1_mortality 1_control mortality which incorporates Abbott’s method (Abbott, 1925) to correct for control mortality. The treatment time, t, is the independent variable and the equation used for S at each temperature is S(T, t)_100e_(kL t)2 (2) This form is obtained by simplifying a form, S_100e_(c_kt)1:a, suggested by earlier workers (King et al., 1979) and used by several authors to model lethal response of fruit flies (Jang, 1986; Laidlaw and Hayes, 1990). The constant ‘c’ is normally small and was omitted, ‘1:a’ ranges from 1.5 to 2.5 and was set near the mid-point of the range so that only the ‘rate’ constant, kL, was retained as a temperature dependent parameter. To simplify notation we drop the explicit reference to the independent variables and write S(T, t)_S.



Experimental facilities and equipment

Eggs were heat-treated in plexiglass tubes (34 mm internal diam. by 50 mm length). Fine material gauze (5.5 strands per mm, 35% open area) inserts were placed into the end of each tube to keep the eggs in the tube while facilitating rapid water movement through the tube. Treatments were applied in a water bath system (Purbeck Limited, New Lynn, Auckland, NZ) consisting of four 25 l water baths. Heater units (Grant, Cambridge, UK Model ZD, temperature accuracy± 0.01°C) on each bath were independently controlled by a laptop computer running Workbench PC for Windows data acquisition and control software (Strawberry Tree, Sunnyvale, CA 94086). The target temperatures were verified for each test using an electronic reference thermometer (Model RT200, Measurements Standards Laboratory of New Zealand, Industrial Research Limited, Wellington, NZ) certified for accuracy under the Measurements Standards Act 1992 by the Measurements Standards Laboratory.

Experimental design

Static temperature lethal heat treatments:

To assess mortality response to constant (static) temperature treatments in the lethal range (> 42°C), B. tryoni eggs were immersed in static temperature water baths at 42, 43, 44, 45, 46, 47 and 48°C for various times (1.5–380 min, depending on temperature). The mortality observed for each temperature, T, at intervals ti gave a direct value for mortality as a function of static temperature treatment time. After heat treatment the eggs were hydro-cooled as described above. Three to six replicates were completed at each immersion temperature. Static temperature conditioning heat treatments In order to determine the effects of exposure to temperatures in the non-lethal range (30–42°C) on the mortality response, B. tryoni eggs were first immersed in a water bath at 32, 34, 36, 38, 40 or 42°C for times ranging from 15 min to 12 h (depending on the temperature bath employed). The tubes containing eggs were removed from the pretreatment bath, drained of hot water and within 2 min simultaneously immersed in a second water bath operating at 46°C. Samples were removed after various exposures (0–70 min) until the lethal time for 99% mortality (LT99) could be established. The samples were then hydro-cooled. Comparison of LT99 for conditioned and nonconditioned eggs provided a quantitative measure of the conditioning as a function of temperature and time of the treatment. The temperatures and exposure durations of the pretreatment were chosen so that conditioning and not mortality would result. This was verified in each test with a second control, which experienced the pretreatment component only. Three to seven replicates were completed at each pretreatment time and temperature combination. Fig. 1 illustrates the procedure schematically for a case where the first group of samples was immersed directly in the 46°C target bath, the second group of samples was pretreated at 38°C for 30 min and then immersed in the target 46°C bath, and a final group of samples was pretreated at 38°C for 60 min before being immersed in the target 46°C bath. Samples were removed at intervals from the 46°C bath, the number of non-hatched eggs determined for each sample and the LT99 times evaluated



Experimental conditions

Immediately before treatment, approximately 200 eggs were washed into each treatment tube with between eight and 14 tubes prepared for each test. Treatment tubes were simultaneously immersed at the start of each test. Immediately following the selected immersion time (1 min to 13 h depending on treatment), individual tubes were removed from the hot water bath, drained and immersed in 25.0±1.0°C water for 2 min (hydro-cooling cycle). Control insects for each treatment were hydro-cooled for a period that exceeded the longest hot water immersion of that treatment by 2 min. Following immersion in 25.0±1.0°C water, the treated and control eggs were washed from each tube onto gauze material and placed on moist filter paper inside petri dishes (9 cm diameter). Each petri dish was sealed with a strip of Parafilm®

to prevent egg desiccation.



Ramped heating:

Although the static temperature treatment results provide an assessment of mortality due to exposure to particular temperatures in the range 30–48°C, it is the effect of the continuously changing temperature during ramped heating that comes closer to addressing the insect mortality response as it occurs in whole fruit. To mimic this, B. tryoni eggs were immersed in a water bath which changed temperature linearly with time from 30 to 46°C in 1, 3, 6, or 9 h (heating rates of 16, 5.3, 2.7 and 1.8°C h-1 respectively). The eggs so treated were then maintained at 46°C until the LT99 value could be established. A comparison of the LT99 for eggs undergoing ramped heating and the LT99s for eggs immersed in 46°C without prior treatment then provided a direct measure of the conditioning due to temperatures and times encountered for a particular ramp. The effect of ramped heating from 25 to 48°C in 1 h (23°C h-1) was also determined. Longer ramp times to a target of 48°C were of limited value because of the high levels of kill at 48°C and temperatures near this target. Three replicate tests were conducted at each ramp. Fig. 2 contains a schematic of the procedure for the smooth linear ramp used in experiments as well as a step-wise ramp which will be used later to illustrate the connection between static and ramped treatments.





Monitoring of critical parameters



Methodology to measure the effectiveness of the treatment

Mortality assessment:

Egg hatch normally begins about 40 h after laying. Treated and control eggs were held at 26±1°C for 3 days to ensure all hatching was complete and then examined for mortality using a binocular microscope 10-40 x magnification). Egg mortality was based on the presence or absence of an exit rupture in the chorion.



Determination of efficacy over a range of critical parameters, where appropriate



Methodology to measure phytotoxicity, when appropriate



Dosimetry system, calibration and accuracy of measurements, if using irradiation



3.2.2 Efficacy data using operational conditions (historical data, may in some cases substitute for the requested information below)

Pest information

Identity of the pest to the appropriate level, life stage, and if a laboratory or field strain was used



Conditions under which the pests are cultured, reared or grown



Biological traits of the pest relevant to the treatment



Method of natural or artificial infestation



Determination of most resistant species/life stage (in the regulated article where appropriate)



Regulated article information

Type of regulated article and intended use



Botanical name for plant or plant product (where applicable)



Conditions of the plant or plant product



Experimental parameters

Level of confidence of laboratory tests provided by the method of statistical analysis and the data supporting that calculation



Experimental facilities and equipment



Experimental design



Experimental conditions



Monitoring of critical parameters



Methodology to measure the effectiveness of the treatment



Determination of efficacy over a range of critical parameters, where appropriate



Methodology to measure phytotoxicity, when appropriate



Dosimetry system, calibration and accuracy of measurements, if using irradiation



Factors that affect the efficacy of the treatment



Monitoring of critical parameters



Special procedures that affect the success of the treatment, if applicable



3.3 Feasibility and applicability (Information should be provided where appropriate on the following items)

Procedure for carrying out the phytosanitary treatment



Cost of typical treatment facility and operational running costs if appropriate



Commercial relevance, including affordability



Extent to which other NPPOs have approved the treatment as a phytosanitary measure



Availability of expertise needed to apply the phytosanitary treatment



Versatility of the phytosanitary treatment



The degree to which the phytosanitary treatment complements other phytosanitary measures



Summary of available information of potential undesirable side-effects



Applicability of treatment with respect to specific regulated article/pest combinations



Technical viability



Phytotoxicity and other effects on the quality of regulated articles, when appropriate



Consideration of the risk of the target organism having or developing resistance to the treatment





Send submissions to:

E-mail: ippc@fao.org Fax: (+39) 06 5705 6347 Mail: IPPC Secretariat (AGPP) (preferred) Food and Agriculture Organization of the UN Viale delle Terme di Caracalla 00153 Rome, Italy

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