Short communication
Phosphine as a replacement for methyl bromide for
postharvest disinfestation of citrus
P. Williams * , G. Hepworth, F. Goubran, M. Muhunthan, K. Dunn
Agriculture Victoria,Institute for Horticultural De6elopment,Pri6ate Bag15,South Eastern Mail Centre,Victoria3176,Australia
Received 17 September 1999; accepted 3 March 2000
Abstract
Methyl bromide is the only fumigant registered for disinfestation of citrus in Australia. However, it has been identified as an ozone-depleting chemical and its use is being restricted in accordance with an international agreement. The only alternative to a 2-h methyl bromide fumigation is cold treatment at 1°C for 16 days, and a more rapid alternative is desirable. The phosphine cylinder gas formulation ECO2FUME (phosphine with CO2as a carrier gas)
was recently registered for 15-h fumigation of cut flowers. This treatment was used as a basis for experimental fumigations of oranges infested with larvae of Queensland fruit fly,Bactrocera tryoniin a 900-l chamber. Uninfested oranges were included in all fumigations to check for possible adverse effects. A 16-h fumigation at 20°C with an initial phosphine concentration of 0.98 g m−3resulted in 96.4% mortality of fly larvae. This was a promising result
but the mortality achieved was insufficient to meet mortality requirements for interstate (99.5%) or export trade (99.9%). Exposure times, temperatures and phosphine concentrations were all increased in subsequent fumigations. In the final series of fumigations with export grade Washington Navel oranges the exposure time was 48 h, thermostat settings for the chamber heater were 23 or 25°C, initial phosphine concentrations were 1.67 g m−3 and the
concentrations were topped up to 0.7 g m−3after 24 h. No adverse effects on the oranges were observed, and a
mortality of 99.998% was achieved with \48 000 larvae exposed. This would meet requirements for interstate trade and possibly also some international trade, particularly if incorporated as part of a systems approach. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Phosphine; Postharvest; Disinfestation; Citrus; Fruit fly
www.elsevier.com/locate/postharvbio
1. Introduction
Citrus infested with larvae of Queensland fruit fly (Qfly), Bactrocera tryoni (Froggatt), is unac-ceptable in the marketplace. Two treatments are currently registered for control of the fruit fly * Corresponding author. Tel.:+61-3-92109222; fax:+
61-3-98003521.
E-mail address:[email protected] (P. Williams)
P.Williams et al./Posthar6est Biology and Technology19 (2000) 193 – 199 194
when fruit is sent interstate or exported. They are fumigated with methyl bromide for 2 h or cold treatment at 1°C for 16 days. Methyl bromide can damage some citrus, e.g. mandarin oranges, and it has been identified as an ozone-depleting chemical with its use is being restricted in accordance with an international agreement, the Montreal Proto-col (Anonymous 1998). Currently there are no restrictions on use of methyl bromide as a com-modity treatment for pre-shipment and quaran-tine purposes, but in the future restrictions may be imposed, with the deadline for phase-out now advanced to 2005.
Cold treatment for 16 days places time con-straints on handling of fruit, and some types of citrus, e.g. oranges and lemons, may suffer chill-ing injury (Goubran, 1989). Handlchill-ing constraints are a particular problem for growers when out-breaks of Qfly occur in areas generally free of the fly. Exclusion zones of 15 km diameter centred on the outbreak site are imposed to make movement of fruit illegal unless it has been subjected to an approved disinfestation treatment. Many growers have limited cool room space and would be severely disadvantaged if all their fruit had to be cold-treated before it could go to market. Conse-quently development of other alternatives to methyl bromide is considered a high priority re-search objective by the citrus industry (Anony-mous, 1996). Surface treatments with petroleum oils have recently been developed for control of surface pests such as scale insects (Taverner et al., 1998) but they do not provide adequate control of pests that live within the fruit, such as fruit fly larvae.
Phosphine is used extensively as a fumigant for stored produce, having the advantage of leaving no detectable toxic residues in produce following ventilation. However, it has the disadvantage that longer fumigation times are required than for methyl bromide (Bond, 1984). Traditionally, phosphine fumigation is carried out using solid formulations of aluminium and magnesium phos-phide from which the gas is gradually evolved. Some work has been done with these formula-tions, placing them in water to accelerate the rate of release of phosphine. However, exposure times were limited to 4 h (to be competitive with
methyl bromide), concentrations were similar to those used in grain and the treatments were not sufficiently effective against Qfly (R. Corcoran, personal communication, 1997). Successful fumi-gation of fruit, including tomatoes and grapefruit infested with Oriental, Mediterranean and Caribbean fruit flies, using solid phosphine gener-ating formulations, has been reported (Seo et al., 1979; Hatton et al., 1982). Exposure periods for these fumigations were much longer (up to 4 days) than for methyl bromide.
During the 1980s, a cylinder gas formulation of phosphine, Phosfume®
(2% phosphine with car-bon dioxide as a carrier gas) was developed (Winks and Russell, 1994), eliminating problems of powder residues associated with solid formula-tions and enabling fumigation times to be re-duced. Phosfume®, recently renamed
ECO2FUME®, has been used successfully in
ex-periments to control several important pests of Australian wildflowers for export using phosphine concentrations of up to 1 g m−3 and exposure
periods of up to 16 h (Muhunthan et al., 1997; Williams and Muhunthan, 1998).
The objective of this study was to assess the potential for use of the phosphine formulation ECO2FUME
®
for disinfestation of citrus infested with Qfly larvae.
2. Materials and methods
2.1. Infesting and handling oranges
that might have crawled into the boxes. At KTRI the oranges were held in a quarantine insectary at 26°C (92°C) and 40 – 70% RH to provide favourable conditions for the development of Qflies. The ‘control’ oranges were held in a refrigerator at 11°C. Oranges used were Leng Navels from Mildura, Victoria and Washington Navels from Yanco and Forbes, New South Wales.
2.2. Fumigating oranges
Cartons of oranges were fumigated with ECO2FUME
® in a 900-l chamber fitted with a
heater and a gas recirculation and exhaust fan. Phosphine concentrations were determined using gas detector tubes (Dra¨ger®) shortly after
intro-duction of ECO2FUME ®
, and again just before the end of each fumigation. In fumigations lasting longer than 24 h, phosphine concentrations were often also determined after half the fumigation time had elapsed. On some occasions additional phosphine was introduced at this time and the boosted concentration measured.
The first fumigations of Leng Navel oranges were based on a schedule developed for wild flowers (Muhunthan et al., 1997). Initial phos-phine concentrations ranging from 0.94 to 1.36 g m−3were used with exposure times ranging from
16 to 24 h. Average temperatures during fumiga-tion ranged between 20 and 23°C. The heater thermostat in the fumigation chamber was set at 20°C, but often the temperature in the chamber was above 20°C, because of the higher initial temperature of the oranges and higher ambient temperatures. Temperatures were recorded every 2 min by two Tinytag®loggers with probes placed
amongst infested oranges. In each fumigation, three or four cartons each containing 80 – 96 in-fested oranges were used together with a carton of 20 uninfested oranges. In fumigations of Wash-ington Navel oranges, exposure time was in-creased up to 48 h and initial phosphine concentrations of up to 1.8 g m−3were used. The
thermostat was set to give 20, 23 or 25°C and temperatures within an infested orange were mea-sured by a probe.
2.3. Assessing effecti6eness of fumigations
Four groups of ten infested oranges were sam-pled from the cartons. Each group was placed in a plastic box 580 mm×380 mm×165 mm with a perforated base, which fitted on top of a box of similar dimensions with a solid base, covered with fine grade vermiculite. Mature larvae left the fruit, passed through the perforations and pupated in the vermiculite. Each pair of boxes was enclosed in a terylene voile bag to prevent escapes. About 10 days later the vermiculite was sieved for pu-paria. If larvae were found, sieving was repeated a few days later. The number of puparia formed was used to estimate the number of larvae infest-ing oranges that were fumigated.
Following each fumigation oranges were taken from the cartons and placed in similar plastic boxes to those used for the unfumigated, infested oranges. After the last puparia from the boxes of unfumigated infested oranges were collected, the boxes of fumigated oranges were sieved for pu-paria. Then the oranges, vermiculite and puparia were autoclaved. A few puparia were kept to see if they would complete development to the adult stage, before being killed. Effectiveness of the fumigations was assessed by estimating percentage mortality using the number of puparia produced compared with the estimated number of larvae exposed. The estimate of larval numbers was based on the average number of pupae per orange produced from batches of unfumigated infested oranges. Lower 95% confidence limits for mortal-ity were found using exact calculations based on the binomial distribution. This is the method of calculation used by Couey and Chew (1986) to relate the number of insects exposed to mortality and probit values.
2.4. Assessing effects of fumigations on oranges
P.Williams et al./Posthar6est Biology and Technology19 (2000) 193 – 199 196
3. Results and discussion
3.1. Effecti6eness of fumigations on fruit fly lar6ae
In fumigations with Leng Navel oranges the thermostat on the fumigation chamber was set to give a temperature of 20°C, initial phosphine con-centrations ranged from 0.94 to 1.36 g m−3 and
exposure times ranged from 16 to 24 h (Table 1). When the oranges were transferred from the car-tons in which they were fumigated to the incuba-tion boxes, dead larvae were observed on many of the oranges. The number of larvae that emerged from these oranges to pupate was far less than the estimated number of larvae exposed (based on pupae produced from the infested unfumigated oranges) (Table 1). While the furnigations showed that phosphine had promise as a control measure, the number of pupae produced was considered commercially unacceptable for export consign-ments of citrus, for which a minimum mortality level of 99.99% is generally required. For inter-state trade the minimum level of mortality ac-cepted is 99.5%. Although this level was exceeded in the best fumigation (Table 1) it was considered that using an initial phosphine concentration of
1.3 g m−3 or more and an exposure time of \24 h would give greater reliability to the treat-ment. Increasing exposure time is known to be very effective in improving efficiency and reliabil-ity of phosphine fumigations (Winks, 1987).
Washington Navel oranges were used in two subsequent series of fumigations. In the first of these the thermostat was set on 20°C, the
maxi-mum initial dosage of phosphine was 1.46 g m−3
and exposure times were 40 and 48 h. In two of the 48-h fumigations phosphine concentrations were measured after 24 h and then topped up to 0.7 g m−3. There appeared to be advantages in
using a 48-h rather than a 40-h exposure period (Table 2). Results obtained in the 48-h tions were better than for any previous fumiga-tions and would certainly be acceptable for interstate trade and export to some countries, as all these fumigations gave mortality levels exceed-ing 99.9%. Further improvement was possible by increasing either the exposure time or the temper-ature of the fumigation. It was decided to increase the temperature rather than the exposure time, because it was considered likely to be more ac-ceptable to industry. Thermostat settings were increased to 23 and 25°C.
Two fumigations were carried out with the thermostat set at 23°C and two at 25°C. In the first fumigation, phosphine concentrations were measured at the beginning and end of the expo-sure time (Table 2). The final concentration of phosphine (0.14 g m−3) was considered lower
than desirable, particularly because it could have been low for many hours. To overcome this prob-lem in subsequent fumigations, concentrations were checked after 24 h and topped up to 0.58 or 0.70 g m−3. Only one pupa, from which the adult
fly did not emerge, was produced from all the fumigations with thermostat settings of 23 or 25°C in which initial phosphine concentrations were 1.67 g m−3 and the concentrations were
topped up after 24 h. The percentage mortalities achieved and lower confidence limits shown in
Table 1
Fumigations of Leng Navel oranges infested with Queensland fruit fly larvae
No. of pupae Estimated no. of
Temperature Exposure time Phosphine concentration Mortality (%) fly larvae
21 8460 53 99.37
20 20 1.08 0.81 12 375 488 96.06
23 20 1.36 0.58 12 874 201 98.44
99.83
24 1.26 0.49 9248 16
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Table 2
Fumigations of Washington navel oranges infested with Queensland fruit fly larvae
Exposure time
Temperature Phosphine concentration (g m−3) Estimated no. of No. of pupae Mortality (%) 95% lower confidence
fly larvae produced limit
(h) (°C)
Initial Middle Final
23 99.810
0.35 12 112
20 40 1.13
2 99.983
20 48 1.25 0.31–0.70 0.36 12 112
8 99.946
14 896 0.35
20 48 1.46 0.54–0.70
0.14 14 144 0 100
48
20 1.39
5 99.974 99.909
19 552
1.46 0.14
23 48
19 552
48 1.67 0.29–0.58 0.10 0 100 99.985
23
0.14 16 256 1 99.994 99.971
25 48 1.67 0.21–0.70
25 48 1.67 0.35–0.70 0.14 12 960 0 100 99.977
1 99.997 99.984
29 216
Both 25 48 1.67 0.70 0.14
P.Williams et al./Posthar6est Biology and Technology19 (2000) 193 – 199 198
Table 2 indicate that the schedule used for these fumigations would be reliable for interstate trade and possibly also for some international trade. particularly if incorporated as part of a systems approach integrating use of control measures, such as baiting, with monitoring and timing of harvest to minimise the chance of cit-rus being infested. Temperatures within oranges and cartons were between 23 and 27°C, except in one fumigation where the carton temperature rose to above 30°C, probably because of mould-ing and rottmould-ing oranges.
3.2. Effects of fumigations on oranges
The Leng Navel oranges maintained their good condition following fumigation. There was no evidence of damage to the rind, and taste and juiciness were unaffected. A similar result was obtained with the Washington Navel or-anges, with the exception of one fumigation. Fu-migated oranges were similar to the unfuFu-migated ones in appearance, juiciness and taste, both on day 1 after fumigation and after storage for a further 4 weeks, when they were all considered commercially acceptable. The exception was the final 48-h fumigation in the first series of fumi-gations with Washington Navels. There was a peculiar smell emanating from the carton of un-infested oranges, and as well the oranges them-selves had a noticeably bitter taste. The cartons of infested oranges in this fumigation contained many oranges with extensive moulding, mainly from green mould (Penicillium digitatum). These cartons had a similar smell to that detected in the carton of uninfested oranges, so the off-odour and bitter taste probably resulted from contamination from the cartons of infested or-anges rather than from the fumigant.
4. Conclusions
This study has demonstrated that phosphine has potential for use as a replacement for methyl bromide for disinfestation of citrus from Qfly. Further work needs to be done to develop means of utilising the fumigation technique by
the citrus industry. It is anticipated that, as with grain fumigations, there will be no problems with residues, but this needs to be demon-strated.
Acknowledgements
This work was conducted with support from the Horticultural Research and Development Corporation (HRDC), the Australian Citrus In-dustry, Agriculture Victoria, NSW Agriculture, Gosford and BOC Gases.
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