phosphine resistance in stored-product insects collected from various grain storage facilities in...

Journal of Stored Products Research 40 (2004) 241–249

Phosphine resistance in stored-product insects collected fromvarious grain storage facilities in Morocco

H. Benhalimaa, M.Q. Chaudhryb,*, K.A. Millsb, N.R. Priceb

a Inspection R!egionale du Contr #ole des Semences et Plants, D.P.V.C.T.R.F., B.P. A6., F"es, MoroccobCentral Science Laboratory, Department for Environment, Food and Rural Affairs, Sand Hutton, York Y041 1LZ, UK

Accepted 24 September 2002

Abstract

Despite heavy dependence on phosphine (PH3) for fumigating stored products, the resistance status ofinsect pests in Morocco has never undergone a thorough investigation. Some control failures with PH3 werereported in Morocco, and a previous study showed two field populations of Sitophilus oryzae to be highlyresistant to phosphine.We surveyed phosphine resistance in field populations of three major insect pests of stored wheat in

Morocco. Around 32% of the samples collected at different storage facilities were found to be infested withone or more species of stored-product beetles. First-generation adult beetles, cultured from the fieldsamples, were subjected to a discriminating dose test for phosphine resistance using an FAO method. Theresults indicated that, with the exception of one population of S. oryzae, all samples tested containedphosphine-resistant individuals. Treatments at up to 1.8 gm�3 of phosphine for 20 h, or at 0.18 gm�3 for upto 5 days, indicated that a high degree of resistance was already selected in some of the insect populations.Tests using [32P]-radiolabelled phosphine showed that the mechanism of resistance in the three insect

species tested involved a reduced uptake of the fumigant. The study has highlighted an urgent need forreviewing current fumigation practices in Morocco to ensure effective use of phosphine and avoid furtherselection of resistance.Crown Copyright r 2003 Published by Elsevier Science Ltd. All rights reserved.

Keywords: Phosphine; Resistance; Stored products; Insects; Morocco

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*Corresponding author. Tel.: +44-1904-462584; fax: +44-1904-462252.

E-mail address: [email protected] (M.Q. Chaudhry).

0022-474X/03/$ - see front matter Crown Copyright r 2003 Published by Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0022-474X(03)00012-2

1. Introduction

Food-grains and other commodities are stored in Morocco as part of food security until thenext harvest. Depending on the length of storage, and the efficacy of prophylactic measuresadopted, these commodities may suffer substantial qualitative and quantitative losses due to avariety of pests, predominantly insects (Benhalima, 1988). The main method used for controllinginsect infestations in stored commodities in Morocco is fumigating with phosphine (PH3) gas. Infact, the ease of use and residue-safe nature of phosphine has increased dependence upon thefumigant in Morocco (Anonymous, 1991) and elsewhere in the world (Taylor, 1989; Chaudhry,2000).As with other control agents, long-term use of a single fumigant has the risk that inadequate

treatments could lead to selection of resistance in pest populations. Indeed, phosphine resistancehas already been reported in a number of countries, with very high levels of resistance in someparts of Asia and Africa (Mills, 1983; Taylor and Halliday, 1986; Benhalima, 1988; Taylor, 1989;Sayaboc et al., 1998), pointing to the possibility of future control failures. At least 11 species ofstored-product insects are now known to have developed resistance to phosphine (Chaudhry,2000), which has been linked to selection pressures exerted by repeated ineffective fumigations insituations where phosphine gas was rapidly lost due to leakage (Halliday et al., 1983; Mills, 1983;Tyler et al., 1983). Another likely factor contributing to the spread of resistance is the movementof insects through international trade in commodities. A global survey undertaken by the Foodand Agriculture Organization (FAO) in 1972–1973 indicated that about 10% of the stored-product insect populations sampled in different countries (including Morocco) containedphosphine-resistant individuals (Champ and Dyte, 1976). Since the survey, some control failureswith phosphine have been experienced in Morocco (Boughdad, 1982), which could be due toresistance in insect pests. A later study by Benhalima (1988) showed that two field strains of riceweevil Sitophilus oryzae (L.), collected at a warehouse in Mekn"es, were highly resistant tophosphine. Reports have also suggested that phosphine resistance has been on the increaseworldwide, including the North African and sub-Saharan regions (Zettler, 1997). The continuedefficacy of phosphine is very important for the Moroccan grain storage industry becausetechnology for the application of non-gaseous pesticides is neither available nor feasible forbagged stored grains.The present studies were aimed at investigating the resistance status of insect pests in Morocco,

to help development of appropriate strategies for effective control of infestations in storedcommodities. Preliminary investigations were also carried out to establish whether the mechanismof resistance in these insects involved a reduced uptake of the fumigant as reported in insects fromother countries (Price, 1981; Chaudhry, 1991; Reichmuth, 1994).

2. Materials and methods

A total of 148 samples of wheat grain were collected during June–July 1999 from grain storagefacilities at 19 different locations, well scattered throughout Morocco. At each storage site,around 2 kg wheat was collected from bags at different heights in bag stacks, and floor sweepings.

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These samples were mixed and a subsample of around 200 g was derived for use in theexperiments. Most of the stores visited had been routinely fumigated with phosphine.Each batch of adult insects, isolated from the wheat samples, was kept separately on 200 g

fumigated wheat, and brought to Central Science Laboratory, UK, where further culturing wascarried out on whole wheat [S. oryzae and lesser grain borer Rhyzopertha dominica (F.)] or wheatflour containing 5% yeast [rust-red flour beetle Tribolium castaneum (Herbst)], at 25�C, 70%relative humidity (r.h.). In all toxicity tests, 8–10 week old first generation adults were used.Resistance was tested by exposing a minimum of 50 adult insects to a discriminating dose of

phosphine following the FAO method (FAO, 1975). Unless otherwise indicated, each test wasreplicated at least three times. A laboratory reference (susceptible) strain of each species was alsoincluded in the tests to verify achievement of the discriminating dose. Each insect species wasexposed to a discriminating concentration of phosphine (0.03 gm�3 for R. dominica, 0.04 gm�3

for S. oryzae and T. castaneum) for 20 h at 25�C. After the exposure, insects were briefly aired andthen kept on food for 2 weeks before mortality was assessed. Further tests at higherconcentrations, longer exposure periods, or both, were carried out on some of the insectpopulation samples. Corrected mortality was calculated as [(% mortality in treated insects�%mortality in control insects)/(100�% mortality in control insects)]� 100.Phosphine gas of about 84% purity (remainder CO2) was generated in a gas burette by reacting

a 0.6 g pellet of aluminium phosphide formulation with 5% sulphuric acid. Appropriate amountsof phosphine were applied to insects in 6.2 l gastight desiccators using a gastight syringe. Theconcentration of phosphine in the source burette, and in test desiccators, was determined by gaschromatography using a flame photometric detector, standardised against phosphine in nitrogendetermined chemically by the method of Bruce et al. (1962).[32P]-radiolabelled phosphine was generated by hydrolysing [32P]-magnesium phosphide,

synthesised by reducing [32P]-orthophosphate and magnesium phosphate in the presence ofmagnesium oxide and magnesium powder (Chaudhry, 1991). The [32P]-phosphine generated in theprocess was carried in a stream of dry nitrogen and condensed in a collection tube immersed inliquid nitrogen. After warming up to room temperature, an aliquot of the gaseous mixturecontaining around 4% [32P]-phosphine in nitrogen was injected into 0.8ml of cold 2% HgCl2solution in a Reactivial through a Mininert valve (Pierce and Warriner, UK). The resultingprecipitate of [32P](HgCl)3 was used in determination of the concentration of [

32P]-phosphine gasafter oxidation with 0.2ml bromine water and development of colour with acid-molybdate-Lubrol-W solution and a-naphthyl-amino-sulphonic acid (Chaudhry, 1991). The absorbance ofthe solution was recorded at 660 nm, and the amount of [32P]-phosphine was calculated using arange of orthophosphate standards. An aliquot of the oxidised [32P](HgCl)3 solution was alsoused for counting Cerenkov emission in a Beckman LS6000TA scintillation counter. The specificactivity of [32P]-phosphine was calculated by comparison with [32P]-orthophosphate standards.One population sample each of R. dominica and T. castaneum, and two of S. oryzae, along with

their respective laboratory susceptible strains, were treated with [32P]-phosphine in a gastight flaskat a concentration of 0.7 gm�3 for 5 h at 25�C. Using a gastight syringe, the calculated amount of[32P]-phosphine was transferred to the test container through a rubber septum. At the end of theexposure, flasks were opened inside a fume cupboard to drive off the radioactive gas for 10–15minbefore counting Cerenkov emission as before. The amount of [32P] retained in treated insects afteraeration was determined by comparison with [32P]-orthophosphate standards.

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H. Benhalima et al. / Journal of Stored Products Research 40 (2004) 241–249 243

3. Results

Out of the 148 samples that were collected from 66 co-operative stores, 46 public warehouses,and 36 private grain stores in Morocco, one-third were found to be infested with one or morespecies of stored-product beetles; predominantly S. oryzae, R. dominica, and T. castaneum. Theresults of discriminating dose tests showed that, apart from one population of S. oryzae (22-CA),all of the population samples tested contained phosphine-resistant individuals (Fig. 1). Thenumber of insects that survived the discriminating dose of phosphine further indicated that someof the population samples tested had a high frequency of resistant individuals. For example, thepopulation samples 2-SK, 129-SK-1 and 25-CA showed more than 90% survival at thediscriminating dose of phosphine.We also attempted to determine the levels of resistance in some of the populations by exposing

them to higher concentrations (up to 1.8 gm�3) of phosphine for 20 h. Samples of a few resistantpopulations were also exposed to a fixed concentration of phosphine (0.18 gm�3) for differentlengths of time (up to 5 days). Our results (Tables 1 and 2) indicated that some populationsamples of S. oryzae tested contained highly resistant individuals. For example, the S. oryzae

population (2-SK) contained individuals that could survive a 20-h exposure to 1.8 gm�3. Anotherpopulation sample of R. dominica (129-SK) showed only 80% mortality when exposed to aconcentration of 1 gm�3 for 20 h at 25�C (data not shown).The results of exposure of some population samples to [32P]-radiolabelled phosphine showed

that [32P] uptake by resistant strains of all three species tested was much lower than that bythe corresponding susceptible strains (Fig. 2). Under similar exposure conditions (0.7 gm�3 of[32P]-phosphine for 5 h at 25�C), the lab susceptible strain of T. castaneum absorbed 60.08 mg ofthe gas per g of insect, whereas a resistant strain (17) absorbed only 8.32mg g�1. Similarly, whilst

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Fig. 1. The response of field populations of three stored-product insect pests collected in Morocco to the discriminating

dose of phosphine.


the lab susceptible strain of R. dominica absorbed 47.67 mg g�1, a resistant strain (128-SK)absorbed 2.42mg g�1. The uptake of [32P]-phosphine by the lab susceptible strain of S. oryzae was14.67mg g�1, whilst the two resistant strains tested (2-SK and 104-FE), absorbed only 5.79 and3.99mg g�1, respectively.

4. Discussion

Phosphine is currently the fumigant of choice in Morocco for disinfecting stored commoditiesthat are mainly stored in bags and stacked up inside a storage building. Occasionally, such stacksare also built outside to allow temporary storage of commodities under the cover of heavytarpaulin sheets. The usual application rate of phosphine for fumigating stored commoditiesranges between 4 and 6 g of phosphine per tonne of grain, over exposure periods that rangebetween 5 and 7 days. The number of fumigations also varies from 1 to 2 for a short-term storage,to 4 or 5 for a relatively longer storage. In public warehouses, fumigation of grains is carried out

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Table 1

Percentage mortality of adults of susceptible and field population samples of S. oryzae after exposure to different

concentrations of phosphine for 20 h at 25�C

Sample No. Concentration of phosphine (gm�3)

0.04 0.11 0.20 0.23 0.39 0.48 0.72 0.86 1.27 1.40 1.70 1.80

Susceptible reference 100 n n n n n n n n n n n

2-SK 10.0 n 52.0 n 76.0 n 78.0 n n 82.0 84.0 92.0

30-SL 12.0 n 68.0 n 82.0 n 92.0 n n 96.0 96.0 100

104-FE 11.3 72.0 n 80.0 n 88.0 n 98.0 100 n n n

Figures shown are % corrected mortality.nNot tested.

Table 2

Percentage mortality of adults of susceptible and field population samples of S. oryzae after exposure to 0.18 gm�3

phosphine over different lengths of time at 25�C

Sample No. Exposure period (days)

1 2 3 4 5

Susceptible reference 100 n n n n

2-SK 50 74 92 97 100

30-SL 44 84 96 100 n

31-SL 54 64 n 98 100

100-KE 44 72 90 98 100

136-SK 94 96 100 n n

143-ME 94 100 n n n

Figures shown are % corrected mortality.nNot tested.


under the cover of plastic sheets to avoid a rapid loss of phosphine. However, some whole-storefumigations are also carried out at co-operatives and private grain stores, where facilities forunder-sheet fumigation are not available.Despite an almost total dependence on the use of phosphine in Morocco since the 1970s,

knowledge of the prevalence of resistance in insect pests has been lacking. The results presentedhere (Fig. 1) show that phosphine resistance is apparently widespread in stored-product insects inMorocco. The fact that 50 out of the 51 insect populations collected from different regionscontained resistant individuals, often in high numbers, indicates that fumigations carried out atthe sites in the past had been inadequate. Considering such a high prevalence of resistance, itwould not be surprising if the absence of resistance in one population of S. oryzae, a naturallytolerant species, was due to an even poorer standard of fumigation at the store in question, notreaching the level required for selection of resistance in immature stages. The extent of theproblem was further highlighted by results of the tests carried out at higher doses of phosphine,and over longer exposure periods, which showed very high levels of resistance in some of thepopulations tested. Some individuals of S. oryzae were able to survive exposure to as much as1.8 gm�3 of phosphine for 20 h. The presence of such a high degree of resistance in fieldpopulations indicates that they have been under gross selection pressure with phosphine, probablyfor many years. These findings are alarming and suggest that current fumigation practices inMorocco are largely ineffective, causing selection of resistance in insect pests.Although the biochemical mechanism of phosphine resistance is not fully known, it has been

shown to be associated with an ‘active exclusion’ of the gas (Price, 1984; Chaudhry and Price,1992), which results in a much lower uptake by resistant insects compared with their susceptiblecounterparts (Price, 1981; Nakakita and Kuroda, 1986; Chaudhry, 1991; Reichmuth, 1994). Theresults of the exposure of some insect populations to [32P]-radiolabelled phosphine (Fig. 2)

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Fig. 2. The uptake of [32P] by three species of insects after exposure to 0.7 gm�3 of [32P]-phosphine for 5 h at 25�C.


showed that the mechanism of resistance in all of the three species tested involves a reduceduptake of the fumigant. The results support earlier findings, which indicated the mechanism ofphosphine resistance to be common among different species of stored-product insects and theirpopulations from different geographical locations (Chaudhry, 1991).The insecticidal action of phosphine differs from other fumigants in that longer exposures to

lower concentrations are more effective than shorter exposures at relatively high concentrations ofthe gas (Hole et al., 1976; Chaudhry and Price, 1990). Thus, increasingly higher concentrations ofphosphine alone may not achieve a proportionately greater effect. The results presented here(Tables 1 and 2) also indicate that longer exposures provide a more effective control of resistantinsects compared to a short exposure to relatively higher concentrations of phosphine. The resultsalso suggest that exposure to 0.18 gm�3 of phosphine for 5 days or more should control resistantadults, including the highly resistant ones encountered in this study. Certainly, a full evaluation ofthe levels of resistance in tolerant stages (eggs and pupae) of the most resistant strains, as carriedout by Price and Mills (1988), is required to determine fully effective dosage schedules. This studyhas, however, highlighted the fact that despite the current practice in Morocco of fumigating at4–6 g of phosphine per tonne of grain for 5–7 days, effective control of insect pests is not beingachieved under field conditions. Under such treatment conditions, repeated re-fumigations areineffective, and only contribute to further selection of resistance. It is possible that resistance insome areas has already reached levels that have contributed to the control failures experienced inMorocco (Boughdad, 1982).A number of factors might lead to failure of a fumigation operation but the use of phosphine in

leaky structures is usually the main cause of underdosing and consequent survival of insect pests.It is imperative, therefore, that all whole-store fumigations in Morocco are abandoned, andphosphine is only used by trained personnel, under gas-tight conditions (e.g. in sealed plastic sheetenclosures) and for the longest exposure periods that are practically feasible in field situations.

5. Conclusions

Around one-third of wheat samples collected from different storage facilities in Morocco werefound to be infested with one or more species of insect pests, and almost all of the populationsamples tested contained resistant individuals, some of them highly resistant to phosphine. Theprevalence and frequency of resistant insects indicates widespread past inadequacy of fumigationpractices in Morocco. Tests also showed that the mechanism of resistance involves a reduceduptake of phosphine, and that it may be possible to control resistance through appropriate use ofphosphine. The studies presented here, nevertheless, do not present a complete picture, and thereis an urgent need for a thorough review of storage practices in Morocco to identify and implementimprovements in currently used fumigation methods.

Acknowledgements

We are grateful to the British Council Morocco for funding the project. We are also grateful toDr. Boughdad for his assistance and help in keeping the back-up stocks of insects in Morocco.

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Our particular thanks are due to the Moroccan National Seed Marketing Company (SONACOS)and to co-operatives and private grain holders for allowing collection of wheat samples from theirstorage facilities.

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