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Field validation of phosphine efficacy on the first recorded resistant strains of Sitophilus granarius and Tribolium castaneum from the Czech Republic
Journal of Stored Products Research 81 (2019) 107e113
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Field validation of phosphine efficacy on the first recorded resistant strains of Sitophilus granarius and Tribolium castaneum from the Czech Republic Radek Aulicky, Vaclav Stejskal*, Barbora Frydova Crop Research Institute, Drnovska 507, 16106, Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2018 Received in revised form 7 February 2019 Accepted 8 February 2019 Available online 22 February 2019
This study first estimated the current state of phosphine sensitivity (using a knock-down/KT100/Degesch kit) in Sitophilus granarius (23 strains) and Tribolium castaneum (8 strains) in Czech Republic grain stores. The resistance of S. granarius (21.7% resistant strains; coefficient of resistance KT100 ranged from 0.5 to 2.3 among strains) was substantially lower and less frequent than that of T. castaneum (87.5% resistant strains; coefficient of resistance KT100 ranged from 0.9 to 52.5 among strains). The phosphine efficacy of the laboratory and field (i.e., resistant) pest strains was validated during commercial fumigation when suboptimal tarpaulin sealing resulted in low-concentration phosphine exposure (Ct products ranged from 5.9 to 7.4 g*hr/m3). Although even low-dose fumigation led to 100% adult mortality of both laboratory and field strains of S. granarius and laboratory strains of T. castaneum, the mortality of the field strain of T. castaneum ranged from 47% to 95%. Larval emergence from the fumigated commodity samples with pest eggs was zero or near zero for laboratory strains, while 1.3e6.0 (S. granarius) and 63.7e80.00 (T. castaneum) field-strain larvae emerged per sample (100 g). This study shows that although a high proportion of the tested pest populations were still sensitive, several T. castaneum populations showed an elevated level of resistance that may decrease field fumigation efficacy, especially under suboptimal phosphine dosage conditions. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Red flour beetle Granary beetle Aluminium phosphide Phosphine Resistance Field efficacy Low dosage
1. Introduction There are a limited number of active ingredients used worldwide for the treatment of stored grain against storage pests. The repeated use of a restricted number of pesticide active ingredients is one of the important conditions for resistance selection. Currently phosphine is the most frequently used gas for the fumigation treatment of stored grain. As such, phosphine is particularly vulnerable to resistance evolution. The ability of storage pests to develop tolerance to phosphine was demonstrated under laboratory conditions almost 50 years ago by Monro and Bond (1972). The first cases of field resistance were reported from grain stores in the 1970s (Champ and Dyte, 1976). Since then, the strength and incidence of resistance to phosphine have been on a historical rise, and published evidence of resistance has accumulated for several storage pests and countries (Opit et al., 2012; Daglish et al., 2015).
* Corresponding author. E-mail address: [email protected] (V. Stejskal). https://doi.org/10.1016/j.jspr.2019.02.003 0022-474X/© 2019 Elsevier Ltd. All rights reserved.
Particularly, warm eco-regions are endangered by the occurrence of phosphine resistance (Taylor and Halliday, 1986). For example, in Australia, Nayak et al. (2017) demonstrated an alarming increase in the incidence of strong resistance in Tribolium castaneum Herbst during the period from 2009 to 2013. Recently, Kocak et al. (2015, 2018) showed that the strong resistance of T. castaneum, Sitophilus oryzae L. and Oryzaephilus surinamensis L. to phosphine is common in grain stores in Turkey. Although resistance to phosphine is considered a serious issue, surprisingly, there are very few published studies on the status of phosphine resistance in stored grain pests in European countries (Champ and Dyte, 1976; Aulicky et al., 2015a; Agrafioti et al., 2017). Ignatowicz (2000) discovered Sitophilus granarius L. resistance to phosphine from a Polish grain store belonging to the northern European ecoregion (https://www. worldwildlife.org/ecoregions/pa0405), and recently, Agrafioti et al. (2017) reported the decreased sensitivity of strains of O. surinamensis and Rhyzopertha dominica F. to phosphine from the southern European ecoregion. However, there is no information on the sensitivity changes of any pest species to phosphine from the grain stores in the remaining European ecoregions, including
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Central Europe. This deficiency is considered a problem from the perspective of fumigation practice. Knowledge of the actual level of pest sensitivity/resistance to phosphine is among the serious prerequisites for the optimization of field fumigation protocols because European Union phosphine labels usually contain a condition-dependent range of doses (e.g., 8e12 g/t of commodity) and exposures (e.g., 5e8 days). There are two main types of grain stores in Czech Republic that includes horizontal flat-stores and vertical silo-stores. The extensive national entomological survey (Stejskal et al., 2003) of the Czech grain stores revealed that the pest faunal composition differed between these two types of stores. The pest complex collected in both types of grain stores included 25 species of mites, 8 species of psocids and 23 species of beetles. While some species of key grain pests (i.e. Acarus siro L., Lachesilla pedicularia L., S. oryzae, R. dominica, O. surinamensis and Cryptolestes ferrugineus Stephens) occurred in both types of stores, a higher frequency and abundance of T. castaneum and S. granarius was recorded in horizontal flatstores than in vertical silo-stores. This is important in terms of potential phosphine resistance development in T. castaneum and S. granarius since phosphine fumigation is the only chemical pest control option in horizontal flat-stores, while sprays (containing pyrethroid or organophosphate active ingredients) may be used as phosphine alternatives for direct grain treatment in vertical stores. However, there has been no information available regarding resistance status in these two pest species to phosphine in Czech Republic. The study has to particular goals. The first goal was to conduct the first survey on phosphine sensitivity in various strains of two particular grain beetle species, S. granarius and T. castaneum, originating from Czech grain stores. The second goal was to investigate how the current level of resistance/sensitivity might influence the efficacy of commercial field fumigation based on currently used fumigation protocols. 2. Materials and methods 2.1. Survey in stores and resistance screening Pests and surveys in stores. In 2014e2016, we sampled 86 grain (barley, wheat, rice) stores. Samples were collected by using a spear sampler and sieves. The detailed sampling pattern followed the same methodology as the previous survey of arthropod fauna in Czech grain stores (Stejskal et al., 2015). The study specifically targeted two beetle pests (granary beetle - Sitophilus granarius and red flour beetle - Tribolium castaneum) that currently frequently occur in Czech commercial grain and seed stores (Stejskal et al., 2014, 2015). Pest breeding and preparation for tests. Before testing, we kept and bred the pests in a laboratory for three generations (F3). For the resistance test, 14-day-old adults were used; the temperature was approximately 23e25 C and RH was 65% (±5%) As the control, sensitive Crop Research Institute (CRI) strains (S. granarius and T. castaneum) were used; these strains were kept at the Crop Research Institute (CRI) for many generations without contact with any insecticide. Laboratory resistance tests and statistics. In this study, we used a commercially available standardized Detia Degesch Phosphine Resistance Test Kit (DDPRK; Detia Degesch GmbH, Germany) proposed by (Steuerwald et al., 2006). This test is a modified version of knock-down method originally developed by C. Reichmuth (Reichmuth, 1991) at the Julius Kühn Institute in Berlin, Germany. His testing protocol is based on a simple rule that if adults are still moving normally after 30 min exposure to 1 mg. l1 PH3, then they are classified as a PH3 resistant strain, but if they are knocked down
within 30 min they are classified as susceptible. The DDPRK test is based on the principle that insects that are still moving after 8 min (T. castaneum) or 12 min (S. granarius) under a phosphine concentration of 3000 ppm or higher are considered resistant to phosphine. In contrast to the experimental approach originally proposed by Reichmuth (1991), DDPRK method uses higher concentration and shorter exposure times (Steuerwald et al., 2006). The DDPRK contains a 100 ml syringe, a 2 cannula, 1 cannula with a rubber hose, a 5 l flexible plastic canister, a lid with a septum, 5 2 test kit pellets in a foil pouch, and a diagram and table to determine the dilution and instructions for use. The DDPRK tests were repeated (with 10 adults per testing vial) 20 times for each species and strain. The knock-down time 99% (KT99) results were analysed by a logistic regression knock-down model (c2 test) using the statistical program XLSTAT (Addinsoft, France). Values of knockdown time 100% (KT100), based on the DDPRK test, were calculated as the average values from all replications. The KT100 values were compared among the strains of each species by using one-way ANOVA with the package STATISTICA 12 (StatSoft CR s.r.o.). KT100 values were separated by post hoc Tukey’s HSD test. Before analysis, all data were submitted to normalization of variance. Two resistance coefficients were estimated separately, either for the KT100 parameter (calculated according to the DDPRK) or for the KT99 parameter (calculated by regression model): (i) The resistance coefficient for KT100 was established as the ratio of KT100 for the Czech field-collected strains to the fixed KT100 value reported as the resistance threshold level in the DDPRK protocol (i.e., fixed value KT100 8 min for sensitive strains of T. castaneum and KT100 12 min for sensitive strains of S. granarius). (ii) The resistance coefficient for KT99 was estimated as the ratio of the KT99 of Czech field strains to the KT99 of the CRI-susceptible reference laboratory strains (i.e., T. castaneum CRI-TcLab e KT99 ¼ 6.93 and S. granarius CRI-SgLab e KT99 ¼ 6.94, Table 1 and Table 2). 2.2. Field validation of fumigation efficacy in field and laboratory strains Fumigation protocol and measurements. Because we were interested in real-world commercial fumigation efficacy, the observation and experiments were performed in a small food factory that had previously reported problems with a decreased phosphine fumigation efficacy. Fumigation was executed by a professional subcontracted fumigation company, and we did not influence either fumigation design (sealing) or dosage. We were only allowed (by factory management and in the presence of fumigation company personnel) to insert our biological samples and measure the phosphine concentration. The treated commodity included 16.2 tonnes (18 big-bags) of soybean flakes. Large bags were placed on the floor of the concrete flat store (432 m3) without windows (Fig. 1). Although soya flakes were infested by T. confusum, this species was not included in the study because it was not part of previous resistance screening. The applied dosage was 5 g PH3.t1 at 96 h of exposure. Phosphine was released from 81 aluminium phosphine flat tablets (Gastoxin; Delicia Freyberg, Germany). After application, the large bags were covered with a tarpaulin of thin plastic sheeting (foil) (Fig. 1). The sheeting was not made gastight with sticky tape. The volume under the tarpaulin was 24 m3, yielding a dosage of 5 g PH3.t1 maximum theoretical concentration of 2426.6 ppm. The phosphine concentration was read using €ger, Germany) and plastic liners the X-am 7000 instrument (Dra from 3 positions: 2 under the tarpaulin and 1 outside the tarpaulin (Fig. 1). The temperature (av. 25.8 C; range 24.6e28.8 C) and relative air humidity (av. 51.5%; range 44.5e53.5%) at location No. 2 were measured (Tinytag ULTRA 2, Gemini Data Loggers, United Kingdom) (Fig. 1).
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Table 1 Detia Degesch Phosphine Resistance Test Kit phosphine resistance coefficient for 23 populations of Sitophilus granarius and statistical comparison among strains. Strain n
Susceptibility time period (min)
KT100 averageb (min)
Resistance coefficient KT100
Slope±SE
KT99 (95% CI)
Resistance coefficient KT99
c2
df P
SgLaba SgDe SgPol SgSe SgChc SgHb SgKr SgZd SgZo SgKl SgChl SgAd SgUh SgHo SgCht SgRad SgRak SgBu SgVy
12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
6.6 ± 0.2abc 6.0 ± 0.3a 6.1 ± 0.1 ab 6.2 ± 0.1abc 6.6 ± 0.3abc 7.7 ± 0.2abcd 7.8 ± 0.5abcd 7.8 ± 0.3abcd 7.9 ± 0.1abcd 8.0 ± 0.3abcd 8.1 ± 0.2abcd 8.8 ± 0.3abcd 9.2 ± 0.5abcde 10.1 ± 0.7abcde 10.3 ± 0.2abcde 10.6 ± 2.0abcde 11.0 ± 1.1abcde 11.7 ± 1.2bcde 11.8 ± 0.5cde
0.6 0.5 0.5 0.5 0.6 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.8 0.8 0.9 0.9 0.9 1.0 1.0
8.69 ± 2.73 8.29 ± 2.22 8.54 ± 2.54 5.76 ± 1.61 3.89 ± 1.32 7.80 ± 2.25 2.88 ± 0.91 5.02 ± 1.17 7.42 ± 2.13 2.88 ± 0.94 10.05 ± 3.05 6.21 ± 1.50 6.38 ± 1.49 2.48 ± 0.66 9.63 ± 2.42 2.06 ± 0.63 2.48 ± 0.63 2.45 ± 0.59 2.19 ± 0.65
1.0 0.8 1.0 1.0 1.2 1.0 1.8 1.5 1.2 1.8 1.1 1.4 1.5 2.5 1.6 2.5 2.5 2.9 3.4
34.27 32.68 34.13 21.53 11.44 32.01 12.26 28.24 30.40 11.82 45.86 41.71 55.63 17.94 54.44 13.90 20.42 21.98 12.90
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
SgTr 20 12 SgKz 20 12 SgPou 20 12
13.1 ± 1.1de 13.2 ± 1.8de 14.8 ± 1.6e
1.1 1.1 1.2
2.30 ± 0.55 2.33 ± 0.58 2.00 ± 0.52
3.5 2.9 4.5
21.28 1 <0.0001 21.57 1 <0.0001 17.31 1 <0.0001
SgDo
20 12
28.0 ± 2.4f
2.3
1.51 ± 0.38
14.0
18.02 1 <0.0001
SgPod 20 12
28.1 ± 2.7f
2.3
2.94 ± 0.38
6.93 (5.45e16.34) 5.82 (4.50e11.62) 6.62 (5.18e14.57) 6.76 (4.81e18.82) 8.49 (5.33e64.19) 7.25 (5.65e15.57) 12.73 (7.47e93.94) 10.40 (7.41e23.51) 8.49 (6.52e19.42) 12.27 (7.19e99.59) 7.84 (6.35e16.22) 9.76 (7.44e19.06) 10.10 (7.79e18.57) 17.11 (10.25e69.54) 10.87 (8.99e17.86) 17.45 (9.75e116.20) 17.56 (10.65e64.00) 20.18 (12.23e69.49) 23.60 (12.60 e195.82) 24.54 (14.45e91.39) 20.41 (12.17e75.54) 31.06 (16.90 e165.97) 97.32 (41.80 e1009.13) 88.35 (39.80 e728.55)
12.7
19.56 1 <0.0001
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Bold indicates susceptible laboratory-reference strain. a Standard susceptible population. b KT100 values within a given experiment that have the same lowercase letter are not significantly different (P 0.05; HSD).
Table 2 Detia Degesch Phosphine Resistance Test Kit phosphine resistance coefficient for 8 populations of Tribolium castaneum and the statistical comparison among strains. Strain n
Susceptibility time period (min)
KT100 averageb (min)
Resistance coefficient KT100
Slope±SE
KT99 (95% CL)
Resistance coefficient KT99
c2
df P
TcLaba TcBu TcPo TcAd TcMb TcUn TcPr TcEt TcBo
8 8 8 8 8 8 8 8 8
6.1 ± 0.1a 7.5 ± 0.5a 8.9 ± 0.3a 10.7 ± 0.2a 11.0 ± 0.3a 11.7 ± 0.2a 13.1 ± 0.6a 357.0 ± 0.3b 420.0 ± 18.5c
0.8 0.9 1.1 1.3 1.4 1.5 1.6 44.6 52.5
6.99 ± 1.81 5.48 ± 1.26 8.43 ± 1.99 13.45 ± 3.50 4.22 ± 0.93 8.14 ± 1.92 6.62 ± 1.29 2.32 ± 0.27 1.48 ± 0.18
6.94 (5.21e14.86) 9.04 (6.63e18.54) 10.72 (8.64e18.18) 11.46 (9.86e317.34) 15.87 (10.97e37.06) 12.42 (10.03e20.98) 15.08 (12.09e23.68) 366.44 (225.04e772.48) 1085.17 (531.67 e33116.48)
1.0 1.3 1.5 1.7 2.3 1.8 2.2 52.8 156.4
29.28 35.87 52.33 57.68 29.99 48.90 78.17 133.27 95.41
1 1 1 1 1 1 1 1 1
20 20 20 20 20 20 20 20 20
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Bold indicates susceptible laboratory-reference strain. a Standard susceptible population. b KT100 values within a given experiment that have the same lowercase letter are not significantly different (P 0.05; HSD).
Strains, biological checks, exposure set up. The field efficacy of phosphine was compared for laboratory and field strains of S. granarius and T. castaneum. The laboratory strains (SgLab and TcLab) originated from non-resistant strains maintained at Crop Research. Institute. The field strains were obtained from the resistance screening: the most resistant strains of each tested species (S. granarius; T. castaneum) from the previous screening were selected. The field bioassays of adults and eggs of S. granarius (SgPod) and T. castaneum (TcEt) were performed using plastic containers (volume 125 ml; 52 mm in diameter, 74 mm in height; Gosselin SAS, France) with lids and bottoms covered by Miralontextile mesh (UHELON, polyamid, 9.5139; Silk and Progress s.r.o., Czech Republic) to enable gas penetration. At each of the three locations, a set of 3 bioassay containers was placed that included 50 adults and commodity with pest eggs. To obtain commodity with
eggs, 50 unsexed adults (we assumed a 50% sex-ratio) were placed in containers and left to oviposit (at 25e26 C and 75% RH) for 72 h before fumigation. For each species, different commodities were used: wheat 100 ± 1 g for S. granarius and 80 ± 1 g wheat and 10 ± 1 g oat flakes for T. castaneum. Adults were gently removed shortly before transportation of the vials to the fumigation site from the laboratory. Before sheeting the stacks, vials with biological samples of laboratory sensitive strains and field resistant strains were placed at three positions (Fig. 1): 2 positions under the sheets and 1 position outside the sheets. Each position received 3 replicates from each tested species, strain and stage (i.e., adults or eggs). Efficacy evaluation and statistics. Adult mortality was evaluated immediately after the termination of treatment. The fumigation efficacy on eggs was further monitored at 55 days after the end of exposure for both species. The results were statistically
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fumigated parts of the store outside the tarpaulin indicated poor sealing, likely due to the very thin PVC tarpaulin used. Table 3 summarizes the information on the mortality of adults of the laboratory and field strains of T. castaneum (TcEt) and of S. granarius (SgPod) at the three positions and the control after the fumigation period. The effects of strain and position were significant (ANOVA; F/9,32/¼ 2.935, p ¼ 0.01). As shown in Table 3, even low-dose fumigation (Fig. 1A) resulted in 100% adult mortality of both laboratory and field strains of S. granarius and laboratory strains of T. castaneum. However, the mortality of the field (i.e., resistant) strain of T. castaneum ranged from 47% to 95%. Table 4 presents larval emergence - expressed as number of larvae emerged per container for the field and laboratory strains of T. castaneum and S. granarius at the three positions and the control after the fumigation period. The effects of strain and position were significant (ANOVA; F/9,32/¼ 8.88, p<0.001). Larval emergence from the fumigated commodity samples with pest eggs was zero for T. castaneum or near zero for S. granarius in laboratory strains. Different situations were observed for the field strains of both species, where 1.3e6.0 emerged larvae per 100 g of phosphinefumigated grain for S. granarius and 63.7e80.0 emerged larvae per 100 g of fumigated cereal flakes for T. castaneum. 4. Discussion Fig. 1. Schematic picture of the flat store with treated commodity and the three positions of the bioassays where the phosphine concentration was measured.
processed and evaluated with the statistical program Statistica 12 using two-way ANOVA. Adult mortality and fumigation efficacy on eggs were separated by post hoc Tukey’s HSD test. Before analysis, all data were submitted to normalization of variance (ln (xþ1)). Means (±SE) of untransformed data are reported. 3. Results 3.1. Survey in stores, strain collection and resistance screening Of the 86 grain stores sampled, 27 stores were positive for any level of T. castaneum, S. granarius or both. We collected 23 strains of S. granarius and 8 strains of T. castaneum for resistance testing. The results of the logistic regression knock-down model and ANOVA evaluation of KT100 (based on DDPRK protocol) are summarized in Tables 1 and 2 Significant differences in the KT100 values were noted in the knock-down time between the tested strains for both species, as shown in Table 1 for S. granarius (F ¼ 28.4; df ¼ 23; p < 0.001) and in Table 2 for T. castaneum (F ¼ 721.6; df ¼ 8; p < 0.001). In the pooled data for both species of stored product pest beetles, we found that of all (n ¼ 31) tested field strains, 38.7% (evaluation by KT100) had at least a minimum level of resistance with a coefficient of 1.1 or higher. In the tested strains, the resistance coefficient KT100 ranged from 0.5 to 2.3 for S. granarius (Table 1) and from 0.9 to 52.5 for T. castaneum (Table 2). 3.2. Field validation of fumigation efficacy in field and laboratory strains Fig. 2 shows the course of concentrations, and Tables 3 and 4 show a summary of the Ct products obtained for each measured position (Fig. 1.). The calculated Ct ranged from 5.9 to 7.4 g*h/m3. The maximum concentration achieved under the tarpaulin was 115 ppm after 24 h from the phosphide tablet introduction. The low phosphine concentration reached under the tarpaulin and the relatively high phosphine concentration detected in the non-
This paper presents analyses of the data from the first study in the Middle European ecoregion concerning the differential sensitivity to phosphine insecticide of various strains of two species of stored-product Coleoptera pests collected during a 3-year grain store survey. For the first historical screening of phosphine resistance in Czech stores, the standard knock-down “Detia Degesch Phosphine Resistance Test Kit (DDPRK) (Steuerwald et al., 2006) was used. DDPRK test belongs to a group of so-called quick resistance tests (Reichmuth, 1991; Chen et al., 2015). As the resistance may be determined in less than one day, these approaches shorten the time needed to assay for resistance in comparisons with a standard FAO phosphine resistance test (FAO, 1975). In our work we decided not to employ the original method (Reichmuth, 1991) but the DDPRK version since it is commercially available and therefore the achieved results can be compared with other studies based on this commercial kit (e.g. Agrafioti et al., 2017). However, the results obtained by the DDPRK quick tests are to some extent compatible even with different methodical approaches. Several studies have shown that the knock-down based kit generates results comparable to those of the more labour- and time-demanding FAO-method (FAO, 1975). For example, Cao et al. (1998) documented a strong positive linear correlation between the logLC50 values (obtained by the FAO method) and logKT50 values obtained by the quick Reichmuth (1991) knock-down method on which the DDPRK is based. Recently, the FAO methods (FAO, 1975) and commercial DDPRK were validated by Agrafioti et al. (2017), and similar results regarding the compatibility of both tests were confirmed. Nevertheless, our study is the first to show that even for a single experimental testing procedure (i.e., DDPRK) using various types of coefficients of resistance, the evaluation of identical data may yield some differences. For example, the comparison of S. granarius strains by KT100 (i.e., calculated according to the instructions provided by the DDPRK) showed only 21.7% of strains with increased resistance, but the KT99 (calculated by the standard logistic regression knock-down model) showed a value of 82.6%. We therefore included both coefficients in this study. Nevertheless, irrespective of data interpretation differences, our results consistently demonstrated that although S. granarius was more
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Concentra on (ppm)
140 120 100 80 60 40 20 0 0
24
47.5
72.5
95.5
Exposure me (hours) Posi on no. 1
Posi on no. 2
Posi on no. 3
Fig. 2. Phosphine concentration reached at the three positions (under and outside the sheets) during the course (95,50 h) of field commercial fumigation.
Table 3 Percentage mortality (mean ± SE) per container of adults of laboratory and field strains of Tribolium castaneum and of Sitophilus granarius at the three positions and the control after the fumigation period. Tribolium castaneum
Position no. 1 Position no. 2 Position no. 3 Control
CT - product (g*h/m3)
Sitophilus granarius
Laboratory strain
Field strain
Laboratory strain
Field strain
100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 1.3 ± 0.7a
18.7 ± 2.4b 95.3 ± 2.7c 46.7 ± 5.7bc 1.3 ± 1.3a
100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 2.0 ± 1.2a
100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 2.7 ± 0.7a
3.3 5.9 7.4 0.0
The means followed from the same lowercase letter are not significantly different; HSD test at 0.05.
Table 4 Adult emergence (mean ± SE) per container for the field and laboratory strains of Tribolium castaneum and of Sitophilus granarius at three positions and the control after the fumigation period. Tribolium castaneum
Position no. 1 Position no. 2 Position no. 3 Control
CT - product (g*h/m3)
Sitophilus granarius
Laboratory strain
Field strain
Laboratory strain
Field strain
0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 134.0 ± 44.1d
85.7 ± 29.2cd 63.7 ± 14.6cd 80.0 ± 45.5cd 98.0 ± 48.2cd
4.7 ± 1.3 ab 0.0 ± 0.0a 0.3 ± 0.3a 90.7 ± 12.0cd
17.3 ± 12.4bc 1.3 ± 0.7 ab 6.0 ± 4.0 ab 81.0 ± 20.1cd
3.3 5.9 7.4 0.0
The means followed from the same lowercase letter are not significantly different; HSD test at 0.05.
frequently found in stores than was T. castaneum, its level of phosphine resistance was neither very frequent nor very high. However, a higher level of phosphine resistance was found in several strains of T. castaneum. For example, one of the tested T. castaneum strains was moving after 420 min of continual phosphine exposure; thus, this strain was approximately 52.5 more tolerant than the sensitive laboratory strain, for which movement stops in less than 8 min. This result can be considered a relatively high resistance level since Reichmuth (1991) described resistant insects as those that are still moving after 30 min of exposure to phosphine at 1 ml l1. Monro and Bond (1972) demonstrated that it took more than 28 generations to obtain a phosphine resistance factor exceeding 3 in S. granarius under laboratory conditions. Although it may seem that this species is not prone to quickly acquire phosphine resistance, there is little evidence for this idea under field grain store
conditions. For S. granarius, there are only two published reports on phosphine field resistance, and these reports provide contradictory results. While Ignatowicz (2000) found this species resistant, Kocak et al. (2018) reported that among the tested species and strains collected from Turkey grain stores, S. granarius was far less resistant to phosphine than were other species; e.g., the maximal coefficient of resistance was only 5.4 for S. granarius, while it was 200.5 for S. oryzae and 459.6 for O. surinamensis. Our results seem to stand between those of Kocak et al., (2018) and Ignatowicz (2000), showing a moderately decreased level of phosphine sensitivity in two strains of S. granarius, while the remaining tested strains were sensitive or very sensitive. Although results from more countries are needed to describe the extent or geographical distribution of the resistance of S. granarius, a different situation can be found for T. castaneum, the other species tested in our study. Our results on the sensitivity of T. castaneum to phosphine seem to fit within the
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broadly reported resistance trends from many geographical ecoregions, showing that the frequency of T. castaneum phosphine resistance in strains is increasing and becoming a global issue (e.g., Australia - Daglish et al., 2015; Nayak et al., 2017; North America e Opit et al., 2012; Asia - Koçak et al., 2018). It is important to note that quick knock-down resistance tests are mainly useful for an initial resistance surveys, such as our study. Although they can be effectively used to detect resistance (i.e. presence/absence) they do not serve well to thoroughly measure the strength of resistance. Particularly, they are not currently designed to accurately distinguish between a weak and strong resistance type (Chen et al., 2015). This may be a major drawback of these methods since distinguishing weak from strong resistance may have profound impact on evaluation of fumigation protocols. For example, the published work from USA (Opit et al., 2012) indicates, that it is likely that weakly resistant populations can be controlled with recommended fumigations that hold 200 ppm for at least three days (Phillips et al., 2012), whereas the strongly resistant populations cannot. Currently there is no published information on strong resistance concerning S. granarius. In contrast to previous species, there is a convincing evidence for occurrence of strong resistance to T. castaneum in several geographical regions (Opit et al., 2012; Koçak et al., 2015; and Nayak et al., 2017). Regarding our work, it may by hypothesised - though not explicitly tested with the method used-that at least one of the T. castaneum strains may be strongly resistant to phosphine, while the others are more likely to be weakly resistant. However, this hypothesis has to be verified by the molecular methods (e. g. Chen et al., 2015). As mentioned in the introduction, resistance to phosphine is a serious issue in several geographical areas from which it may spread around the world. From an international perspective, our resistance survey can be considered a useful missing piece of information from the Middle European region that helps to complete the global mosaic picture showing the current geographical phosphine resistance distribution. However, the resistance survey is only the first part of resistance management (Nayak et al., 2017). Agricultural and food industry practice is interested in not only whether resistance is present in a given area but also if and how the current local strength of resistance to phosphine can project into fumigation protocols (i.e. optimization of phosphine dosage and exposure - Meyvaci et al., 2010) and if there is a need for any label adjustments. We therefore compared the efficacy of phosphine laboratory strains under conditions of commercial fumigation. Our concentration measurements showed that for the observed fumigation, the fumigation company used improper plastic sheeting and attachment to the floor, leading to relatively high phosphine concentrations in the store but low phosphine concentrations and Ct products under the sheets. Wang et al. (2006) documented that the type of sheeting material and its thickness and quality of attachment to the floor strongly affect the retention of fumigant under the tarpaulin. We found similar results for the Czech populations. Even very low concentrations and Ct products, reached under the sheets due to poor sealing, were able to control non-resistant adults and egg stadia in both tested species. We recorded zero survival of lowresistance field strains of S. granarius. The high mortality at low concentrations was likely due to high temperatures (25 C) occurring during fumigation. Previously, it was shown in Czech mill fumigation that high temperature may play a significant role in compensation for a lower phosphine dosage, while the highest concentration achieved at the lowest temperature resulted in egg survival (Aulicky et al., 2015a). In our study, we observed high survival (approximately 50%) of substantially more resistant strains of T. castaneum and emergence from eggs. Our experiment thus confirmed the findings of Wang et al. (2006), who showed that the quality of sealing was a more important factor in controlling
Southern China resistant than sensitive strains of storage pests. Although a single fumigation event, executed under poorly sealed conditions, usually results in little population resistance shift, multiple fumigations of leaky structures is the most likely factor that triggers development of stored product pest resistance to phosphine (Collins, 2009). This indicates that the problem of improper sealing not only hampers phosphine efficacy on resistant strains but may even be a cause of resistance selection and occurrence. For example, a recent Australian analysis by Nayak et al. (2017) showed that resistant populations are especially reported from stores showing improper levels of sealing. It was also found that strong resistance development in T. castaneum was more prevalent in unsealed bunkers, a trend that was also previously reported for S. oryzae (Holloway et al., 2016) and R. dominica (Collins et al., 2017). Their hypothesis is that generally in poorly sealed structures, it is difficult to achieve and maintain not only proper concentration but also gas distribution and commodity penetration. As a result, repeated fumigations to overcome residual populations are not uncommon in structures in which patterns are associated with resistance selection. Our results, along with several other studies (Aulicky and Stejskal, (2015b), Collins et al., 2017; Nayak et al., 2015, 2017), imply that the outcome of phosphine fumigation in the field is a relatively complex interplay between three groups of factors: environmental factors (temperature, wind, and humidity), quality of fumigation technological procedure (sealing, proper dosage, even gas distribution, etc.) (Bell, 2014) and level of pest resistance. In commercial phosphine fumigation, various factors can either enforce (compensate) or weaken other factors. It seems that, for example, excellent sealing and high temperature may partially compensate for the negative effects of low gas dosage or the presence of pest resistance. Conclusions and practical implications. The threat to the future use of phosphine fumigants is not only the frequency of resistance but also its potential increase in strength. Our study demonstrates that, although a high proportion of the tested Czech pest populations were still sensitive, several populations (notably T. castaneum) showed alarming levels of phosphine resistance. Such elevated level of resistance may have impact on the practical use of phosphine in Czech Republic. For example, a decreased field phosphine fumigation efficacy on certain populations of T. castaneum may be encountered, especially under suboptimal dosage conditions. The currently used phosphine EU labels include condition-dependent range of doses and exposures. In case that resistant population occurs, the upper marginal values, from the legally registered range of doses and exposures, should be used; and vice versa. Therefore, farms, mills and commodity traders should apply routine resistance screenings before any fumigation and modify fumigation protocol accordingly. The particular fumigation protocol should also take into account a potential occurrence of weak and strong population resistance to phosphine. In future, it even may be required to alter phosphine rates and duration in order to control the strong resistant populations. However, the practice is currently challenging a problem how to quickly and cheaply determine strong resistance. The molecular methods are costly and the currently used knock-down quick resistance tests are not able to distinguish a weak from a strong resistance. It implies that further research in that venue is still urgently needed. Not only the consequences but also the causes of resistance should be addressed. Pest resistance to fumigants has mainly been associated with high fumigation frequencies and improper fumigation practices (Bell, 2014). The long-term battle against resistance should therefore be based on the support and implementation of anti-resistance strategies that should include fumigation training, extension, the penalization of improper application methods and fumigation malpractice (e.g., Aulicky et al., 2015) and the
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introduction of alternative control methods and active ingredients (e.g., Nayak et al., 2016) with the potential to break the selection process for phosphine resistance. Acknowledgements We thank Sarka Tuckova for excellent technical assistance. This study was supported by the Ministry of Agriculture of the Czech Republic, Projects No. RO 0419 and QJ1530373. References €tze, C., Allegra, J., Athanassiou, C.G., 2017. Evaluation Agrafioti, P., Sotiroudas, V., Go of phosphine resistance in stored-product insects from Greece using two assessment methods. In: Trematerra, P., Trnad, S. (Eds.), Proc. of the 11th Int. Conf. on the IOBC-WPRS Bulletin. Ljubljana, vol. 130, p. 95. Aulicky, R., Stejskal, V., Frydova, B., Athanassiou, C.G., 2015. Susceptibility of two strains of the confused flour beetle (Coleoptera: Tenebrionidae) following phosphine structural mill fumigation: effects of concentration, temperature, and flour deposits. J. Econ. Entomol. 108, 2823e2830. Aulicky, R., Stejskal, V., 2015. Efficacy and limitations of phosphine “spot-fumigation” against five Coleoptera species of stored product pests in wheat in a grain store. Plant Protect. Sci. 51, 33e38. Bell, C.H., 2014. A review of insect responses to various encountered in the managed storage environment. J. Stored Prod. Res. 59, 260e274. Cao, Y., Zhang, J.J., Bengston, M., 1998. Studies on a quick method to measure resistance of four strains of Tribolium castaneum (Herbst) to phosphine. In: Jin, Z.X., et al. (Eds.), Stored Product Protection. Proc. Of the 7th Int. Conf. on Stored-Product Protection, PR China, pp. 603e606. Champ, B.R., Dyte, C.E., 1976. Report of the FAO global survey of pesticide susceptibility of stored grain pests. In: FAO Plant Prot. And Prod. Series, No. 5. FAO, Rome. Chen, Z., Schlipalius, D., Opit, G., Subramanyam, B., Phillips, T.W., 2015. Diagnostic molecular markers for phosphine resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica. PLoS One 10 e0121343. Collins, P.J., 2009. Strategy to Manage Resistance to Phosphine in the Australian Grain Industry. An Initiative of the National Working Party on Grain Protection. Cooperative Research Centre for National Plant Biosecurity Project. CRC70096. http://www.graintrade.org.au/sites/default/files/file/Phosphine_Strategy.pdf. Collins, P.J., Falk, M.G., Nayak, M.K., Emery, R.N., Holloway, J.C., 2017. Monitoring resistance to phosphine in the lesser grain borer, Rhyzopertha dominica, in Australia: a national analysis of trends, storage types and geography in relation to resistance detections. J. Stored Prod. Res. 70, 25e36. Daglish, G.J., Nayak, M.K., Pavic, H., Smith, L.W., 2015. Prevalence and potential fitness cost of weak phosphine resistance in Tribolium castaneum (Herbst) in Eastern Australia. J. Stored Prod. Res. 61, 54e58. FAO, 1975. Method No. 16. Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides. Tentative method for adults of some major pest species of stored cereals, with methyl bromide and phosphine. Plant Protect. Bull. FAO. 23, 12e25. Holloway, J.C., Falk, M.G., Emery, R.N., Collins, P.J., Nayak, M.K., 2016. Resistance to phosphine in Sitophilus oryzae in Australia: a national analysis of trends and frequencies over time and geographic spread. J. Stored Prod. Res. 69, 129e137.
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Ignatowicz, S., 2000. Cases of phosphine resistance in the grain weevil, Sitophilus granarius, found in Poland. In: Zuxun, J., et al. (Eds.), Proc. 7th Int. Working Conf. on Stored-product Protection, Beijing, vol. 1. Sichuan Publishing House of Science & Technology, Chengdu, China, pp. 625e630. Koçak, E., Schlipalius, D., Ramandeep, K., Tuck, A., Ebert, P., Collins, P., Yilmaz, A., 2015. Determining phosphine resistance in rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) populations from Turkey. Turkiye Entomoloji Dergisi 39, 129e136. Koçak, E., Yilmaz, A., Alpkent, Y.N., Ertürk, S., 2018. Phosphine resistance to some coleopteran pests in stored grains across Turkey. In: Trematerra, P., Trdan, S. (Eds.), Proc. of the 11th Int. Conf. on the IOBC-WPRS Bulletin. Ljubljana, vol. 130, pp. 303e310. Meyvaci, K.B., Sen, F., Aksoy, U., 2010. Optimization of magnesium phosphide treatment used to control major dried fig storage pests. Horticult. Environ. Biotechnol. 51, 33e38. Monro, H.A.U., Bond, E.J., 1972. Resistance of a laboratory strain of Sitophilus granarius (rkL) (Coleoptera, Curculionidae) to phosphine. J. Stored Prod. Res. 8, 199e207. Nayak, M.K., Daglish, G.J., Phillips, T.W., 2015. Managing resistance to chemical treatments in stored products pests. Stewart Postharvest Rev 11, 1e6. Nayak, M.K., Jagadeesan, R., Kaur, R., Daglish, G.J., Reid, R., Pavic, H., Smith, L.W., Collins, P.J., 2016. Use of sulfuryl fluoride in the management of strongly phosphine-resistant insect pest populations in bulk grain storages in Australia. J. Grain Storage Res. 16, 100e107. Nayak, M.K., Falk, M.G., Emery, R.N., Collins, P.J., Holloway, J.C., 2017. An analysis of trends, frequencies and factors influencing the development of resistance to phosphine in the red flour beetle Tribolium castaneum (Herbst) in Australia. J. Stored Prod. Res. 72, 35e48. Opit, G.P., Phillips, T.W., Aikins, M.J., Hasan, M.M., 2012. Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma. J. Econ. Entomol. 105, 1107e1114. Phillips, T., Thoms, E., Demark, J., Walse, 2012. Fumigation, chapter 14. In: Stored Product Protection, vols. 156e4. K-State Research and Extension publication, pp. 1e14. Reichmuth, C.A., 1991. Quick test to determine phosphine resistance in stored products research. GASGA Newsletter 15, 14e15. Stejskal, V., Hubert, J., Kucerova, Z., Munzbergova, Z., Lukas, J., Zdarkova, E., 2003. The influence of the type of storage on pest infestation of stored grain in the Czech Republic. Plant Soil Environ. 49, 55e62. Stejskal, V., Aulicky, R., Kucerova, Z., 2014. Pest control strategies and damage potential of seed-infesting pests in the Czech stores e a review. Plant Protect. Sci. 50, 165e173. Stejskal, V., Hubert, J., Aulicky, R., Kucerova, Z., 2015. Overview of present and past and pest-associated risks in stored food and feed products: European perspective. J. Stored Prod. Res. 64, 122e132. Steuerwald, R., Dierks-Lange, H., Schmitt, S., 2006. Rapid bioassay for determining the phosphine tolerance. In: Proc. Of the 9th Int. Working Conf. on StoredProduct Prot., 15-18 October 2006, Campinas, Sao Paolo, BRAPOS, Passo Fundo, RS, Brazil, pp. 306e311. Taylor, R.W.D., Halliday, D., 1986. The geographical spread of resistance to phosphine by Coleopterous pests of stored products. In: Proceedings British Crop Protection Conference, Pests and Diseases, pp. 607e613. Brighton. Wang, D., Collins, P.J., Gao, X., 2006. Optimising indoor phosphine fumigation of paddy rice bag-stacks under sheeting for control of resistant insects. J. Stored Prod. Res. 42, 207e217.
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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Field validation of phosphine efficacy on the first recorded resistant strains of Sitophilus granarius and Tribolium castaneum from the Czech Republic Radek Aulicky, Vaclav Stejskal*, Barbora Frydova Crop Research Institute, Drnovska 507, 16106, Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2018 Received in revised form 7 February 2019 Accepted 8 February 2019 Available online 22 February 2019
This study first estimated the current state of phosphine sensitivity (using a knock-down/KT100/Degesch kit) in Sitophilus granarius (23 strains) and Tribolium castaneum (8 strains) in Czech Republic grain stores. The resistance of S. granarius (21.7% resistant strains; coefficient of resistance KT100 ranged from 0.5 to 2.3 among strains) was substantially lower and less frequent than that of T. castaneum (87.5% resistant strains; coefficient of resistance KT100 ranged from 0.9 to 52.5 among strains). The phosphine efficacy of the laboratory and field (i.e., resistant) pest strains was validated during commercial fumigation when suboptimal tarpaulin sealing resulted in low-concentration phosphine exposure (Ct products ranged from 5.9 to 7.4 g*hr/m3). Although even low-dose fumigation led to 100% adult mortality of both laboratory and field strains of S. granarius and laboratory strains of T. castaneum, the mortality of the field strain of T. castaneum ranged from 47% to 95%. Larval emergence from the fumigated commodity samples with pest eggs was zero or near zero for laboratory strains, while 1.3e6.0 (S. granarius) and 63.7e80.00 (T. castaneum) field-strain larvae emerged per sample (100 g). This study shows that although a high proportion of the tested pest populations were still sensitive, several T. castaneum populations showed an elevated level of resistance that may decrease field fumigation efficacy, especially under suboptimal phosphine dosage conditions. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Red flour beetle Granary beetle Aluminium phosphide Phosphine Resistance Field efficacy Low dosage
1. Introduction There are a limited number of active ingredients used worldwide for the treatment of stored grain against storage pests. The repeated use of a restricted number of pesticide active ingredients is one of the important conditions for resistance selection. Currently phosphine is the most frequently used gas for the fumigation treatment of stored grain. As such, phosphine is particularly vulnerable to resistance evolution. The ability of storage pests to develop tolerance to phosphine was demonstrated under laboratory conditions almost 50 years ago by Monro and Bond (1972). The first cases of field resistance were reported from grain stores in the 1970s (Champ and Dyte, 1976). Since then, the strength and incidence of resistance to phosphine have been on a historical rise, and published evidence of resistance has accumulated for several storage pests and countries (Opit et al., 2012; Daglish et al., 2015).
* Corresponding author. E-mail address: [email protected] (V. Stejskal). https://doi.org/10.1016/j.jspr.2019.02.003 0022-474X/© 2019 Elsevier Ltd. All rights reserved.
Particularly, warm eco-regions are endangered by the occurrence of phosphine resistance (Taylor and Halliday, 1986). For example, in Australia, Nayak et al. (2017) demonstrated an alarming increase in the incidence of strong resistance in Tribolium castaneum Herbst during the period from 2009 to 2013. Recently, Kocak et al. (2015, 2018) showed that the strong resistance of T. castaneum, Sitophilus oryzae L. and Oryzaephilus surinamensis L. to phosphine is common in grain stores in Turkey. Although resistance to phosphine is considered a serious issue, surprisingly, there are very few published studies on the status of phosphine resistance in stored grain pests in European countries (Champ and Dyte, 1976; Aulicky et al., 2015a; Agrafioti et al., 2017). Ignatowicz (2000) discovered Sitophilus granarius L. resistance to phosphine from a Polish grain store belonging to the northern European ecoregion (https://www. worldwildlife.org/ecoregions/pa0405), and recently, Agrafioti et al. (2017) reported the decreased sensitivity of strains of O. surinamensis and Rhyzopertha dominica F. to phosphine from the southern European ecoregion. However, there is no information on the sensitivity changes of any pest species to phosphine from the grain stores in the remaining European ecoregions, including
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Central Europe. This deficiency is considered a problem from the perspective of fumigation practice. Knowledge of the actual level of pest sensitivity/resistance to phosphine is among the serious prerequisites for the optimization of field fumigation protocols because European Union phosphine labels usually contain a condition-dependent range of doses (e.g., 8e12 g/t of commodity) and exposures (e.g., 5e8 days). There are two main types of grain stores in Czech Republic that includes horizontal flat-stores and vertical silo-stores. The extensive national entomological survey (Stejskal et al., 2003) of the Czech grain stores revealed that the pest faunal composition differed between these two types of stores. The pest complex collected in both types of grain stores included 25 species of mites, 8 species of psocids and 23 species of beetles. While some species of key grain pests (i.e. Acarus siro L., Lachesilla pedicularia L., S. oryzae, R. dominica, O. surinamensis and Cryptolestes ferrugineus Stephens) occurred in both types of stores, a higher frequency and abundance of T. castaneum and S. granarius was recorded in horizontal flatstores than in vertical silo-stores. This is important in terms of potential phosphine resistance development in T. castaneum and S. granarius since phosphine fumigation is the only chemical pest control option in horizontal flat-stores, while sprays (containing pyrethroid or organophosphate active ingredients) may be used as phosphine alternatives for direct grain treatment in vertical stores. However, there has been no information available regarding resistance status in these two pest species to phosphine in Czech Republic. The study has to particular goals. The first goal was to conduct the first survey on phosphine sensitivity in various strains of two particular grain beetle species, S. granarius and T. castaneum, originating from Czech grain stores. The second goal was to investigate how the current level of resistance/sensitivity might influence the efficacy of commercial field fumigation based on currently used fumigation protocols. 2. Materials and methods 2.1. Survey in stores and resistance screening Pests and surveys in stores. In 2014e2016, we sampled 86 grain (barley, wheat, rice) stores. Samples were collected by using a spear sampler and sieves. The detailed sampling pattern followed the same methodology as the previous survey of arthropod fauna in Czech grain stores (Stejskal et al., 2015). The study specifically targeted two beetle pests (granary beetle - Sitophilus granarius and red flour beetle - Tribolium castaneum) that currently frequently occur in Czech commercial grain and seed stores (Stejskal et al., 2014, 2015). Pest breeding and preparation for tests. Before testing, we kept and bred the pests in a laboratory for three generations (F3). For the resistance test, 14-day-old adults were used; the temperature was approximately 23e25 C and RH was 65% (±5%) As the control, sensitive Crop Research Institute (CRI) strains (S. granarius and T. castaneum) were used; these strains were kept at the Crop Research Institute (CRI) for many generations without contact with any insecticide. Laboratory resistance tests and statistics. In this study, we used a commercially available standardized Detia Degesch Phosphine Resistance Test Kit (DDPRK; Detia Degesch GmbH, Germany) proposed by (Steuerwald et al., 2006). This test is a modified version of knock-down method originally developed by C. Reichmuth (Reichmuth, 1991) at the Julius Kühn Institute in Berlin, Germany. His testing protocol is based on a simple rule that if adults are still moving normally after 30 min exposure to 1 mg. l1 PH3, then they are classified as a PH3 resistant strain, but if they are knocked down
within 30 min they are classified as susceptible. The DDPRK test is based on the principle that insects that are still moving after 8 min (T. castaneum) or 12 min (S. granarius) under a phosphine concentration of 3000 ppm or higher are considered resistant to phosphine. In contrast to the experimental approach originally proposed by Reichmuth (1991), DDPRK method uses higher concentration and shorter exposure times (Steuerwald et al., 2006). The DDPRK contains a 100 ml syringe, a 2 cannula, 1 cannula with a rubber hose, a 5 l flexible plastic canister, a lid with a septum, 5 2 test kit pellets in a foil pouch, and a diagram and table to determine the dilution and instructions for use. The DDPRK tests were repeated (with 10 adults per testing vial) 20 times for each species and strain. The knock-down time 99% (KT99) results were analysed by a logistic regression knock-down model (c2 test) using the statistical program XLSTAT (Addinsoft, France). Values of knockdown time 100% (KT100), based on the DDPRK test, were calculated as the average values from all replications. The KT100 values were compared among the strains of each species by using one-way ANOVA with the package STATISTICA 12 (StatSoft CR s.r.o.). KT100 values were separated by post hoc Tukey’s HSD test. Before analysis, all data were submitted to normalization of variance. Two resistance coefficients were estimated separately, either for the KT100 parameter (calculated according to the DDPRK) or for the KT99 parameter (calculated by regression model): (i) The resistance coefficient for KT100 was established as the ratio of KT100 for the Czech field-collected strains to the fixed KT100 value reported as the resistance threshold level in the DDPRK protocol (i.e., fixed value KT100 8 min for sensitive strains of T. castaneum and KT100 12 min for sensitive strains of S. granarius). (ii) The resistance coefficient for KT99 was estimated as the ratio of the KT99 of Czech field strains to the KT99 of the CRI-susceptible reference laboratory strains (i.e., T. castaneum CRI-TcLab e KT99 ¼ 6.93 and S. granarius CRI-SgLab e KT99 ¼ 6.94, Table 1 and Table 2). 2.2. Field validation of fumigation efficacy in field and laboratory strains Fumigation protocol and measurements. Because we were interested in real-world commercial fumigation efficacy, the observation and experiments were performed in a small food factory that had previously reported problems with a decreased phosphine fumigation efficacy. Fumigation was executed by a professional subcontracted fumigation company, and we did not influence either fumigation design (sealing) or dosage. We were only allowed (by factory management and in the presence of fumigation company personnel) to insert our biological samples and measure the phosphine concentration. The treated commodity included 16.2 tonnes (18 big-bags) of soybean flakes. Large bags were placed on the floor of the concrete flat store (432 m3) without windows (Fig. 1). Although soya flakes were infested by T. confusum, this species was not included in the study because it was not part of previous resistance screening. The applied dosage was 5 g PH3.t1 at 96 h of exposure. Phosphine was released from 81 aluminium phosphine flat tablets (Gastoxin; Delicia Freyberg, Germany). After application, the large bags were covered with a tarpaulin of thin plastic sheeting (foil) (Fig. 1). The sheeting was not made gastight with sticky tape. The volume under the tarpaulin was 24 m3, yielding a dosage of 5 g PH3.t1 maximum theoretical concentration of 2426.6 ppm. The phosphine concentration was read using €ger, Germany) and plastic liners the X-am 7000 instrument (Dra from 3 positions: 2 under the tarpaulin and 1 outside the tarpaulin (Fig. 1). The temperature (av. 25.8 C; range 24.6e28.8 C) and relative air humidity (av. 51.5%; range 44.5e53.5%) at location No. 2 were measured (Tinytag ULTRA 2, Gemini Data Loggers, United Kingdom) (Fig. 1).
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Table 1 Detia Degesch Phosphine Resistance Test Kit phosphine resistance coefficient for 23 populations of Sitophilus granarius and statistical comparison among strains. Strain n
Susceptibility time period (min)
KT100 averageb (min)
Resistance coefficient KT100
Slope±SE
KT99 (95% CI)
Resistance coefficient KT99
c2
df P
SgLaba SgDe SgPol SgSe SgChc SgHb SgKr SgZd SgZo SgKl SgChl SgAd SgUh SgHo SgCht SgRad SgRak SgBu SgVy
12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
6.6 ± 0.2abc 6.0 ± 0.3a 6.1 ± 0.1 ab 6.2 ± 0.1abc 6.6 ± 0.3abc 7.7 ± 0.2abcd 7.8 ± 0.5abcd 7.8 ± 0.3abcd 7.9 ± 0.1abcd 8.0 ± 0.3abcd 8.1 ± 0.2abcd 8.8 ± 0.3abcd 9.2 ± 0.5abcde 10.1 ± 0.7abcde 10.3 ± 0.2abcde 10.6 ± 2.0abcde 11.0 ± 1.1abcde 11.7 ± 1.2bcde 11.8 ± 0.5cde
0.6 0.5 0.5 0.5 0.6 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.8 0.8 0.9 0.9 0.9 1.0 1.0
8.69 ± 2.73 8.29 ± 2.22 8.54 ± 2.54 5.76 ± 1.61 3.89 ± 1.32 7.80 ± 2.25 2.88 ± 0.91 5.02 ± 1.17 7.42 ± 2.13 2.88 ± 0.94 10.05 ± 3.05 6.21 ± 1.50 6.38 ± 1.49 2.48 ± 0.66 9.63 ± 2.42 2.06 ± 0.63 2.48 ± 0.63 2.45 ± 0.59 2.19 ± 0.65
1.0 0.8 1.0 1.0 1.2 1.0 1.8 1.5 1.2 1.8 1.1 1.4 1.5 2.5 1.6 2.5 2.5 2.9 3.4
34.27 32.68 34.13 21.53 11.44 32.01 12.26 28.24 30.40 11.82 45.86 41.71 55.63 17.94 54.44 13.90 20.42 21.98 12.90
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
SgTr 20 12 SgKz 20 12 SgPou 20 12
13.1 ± 1.1de 13.2 ± 1.8de 14.8 ± 1.6e
1.1 1.1 1.2
2.30 ± 0.55 2.33 ± 0.58 2.00 ± 0.52
3.5 2.9 4.5
21.28 1 <0.0001 21.57 1 <0.0001 17.31 1 <0.0001
SgDo
20 12
28.0 ± 2.4f
2.3
1.51 ± 0.38
14.0
18.02 1 <0.0001
SgPod 20 12
28.1 ± 2.7f
2.3
2.94 ± 0.38
6.93 (5.45e16.34) 5.82 (4.50e11.62) 6.62 (5.18e14.57) 6.76 (4.81e18.82) 8.49 (5.33e64.19) 7.25 (5.65e15.57) 12.73 (7.47e93.94) 10.40 (7.41e23.51) 8.49 (6.52e19.42) 12.27 (7.19e99.59) 7.84 (6.35e16.22) 9.76 (7.44e19.06) 10.10 (7.79e18.57) 17.11 (10.25e69.54) 10.87 (8.99e17.86) 17.45 (9.75e116.20) 17.56 (10.65e64.00) 20.18 (12.23e69.49) 23.60 (12.60 e195.82) 24.54 (14.45e91.39) 20.41 (12.17e75.54) 31.06 (16.90 e165.97) 97.32 (41.80 e1009.13) 88.35 (39.80 e728.55)
12.7
19.56 1 <0.0001
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Bold indicates susceptible laboratory-reference strain. a Standard susceptible population. b KT100 values within a given experiment that have the same lowercase letter are not significantly different (P 0.05; HSD).
Table 2 Detia Degesch Phosphine Resistance Test Kit phosphine resistance coefficient for 8 populations of Tribolium castaneum and the statistical comparison among strains. Strain n
Susceptibility time period (min)
KT100 averageb (min)
Resistance coefficient KT100
Slope±SE
KT99 (95% CL)
Resistance coefficient KT99
c2
df P
TcLaba TcBu TcPo TcAd TcMb TcUn TcPr TcEt TcBo
8 8 8 8 8 8 8 8 8
6.1 ± 0.1a 7.5 ± 0.5a 8.9 ± 0.3a 10.7 ± 0.2a 11.0 ± 0.3a 11.7 ± 0.2a 13.1 ± 0.6a 357.0 ± 0.3b 420.0 ± 18.5c
0.8 0.9 1.1 1.3 1.4 1.5 1.6 44.6 52.5
6.99 ± 1.81 5.48 ± 1.26 8.43 ± 1.99 13.45 ± 3.50 4.22 ± 0.93 8.14 ± 1.92 6.62 ± 1.29 2.32 ± 0.27 1.48 ± 0.18
6.94 (5.21e14.86) 9.04 (6.63e18.54) 10.72 (8.64e18.18) 11.46 (9.86e317.34) 15.87 (10.97e37.06) 12.42 (10.03e20.98) 15.08 (12.09e23.68) 366.44 (225.04e772.48) 1085.17 (531.67 e33116.48)
1.0 1.3 1.5 1.7 2.3 1.8 2.2 52.8 156.4
29.28 35.87 52.33 57.68 29.99 48.90 78.17 133.27 95.41
1 1 1 1 1 1 1 1 1
20 20 20 20 20 20 20 20 20
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Bold indicates susceptible laboratory-reference strain. a Standard susceptible population. b KT100 values within a given experiment that have the same lowercase letter are not significantly different (P 0.05; HSD).
Strains, biological checks, exposure set up. The field efficacy of phosphine was compared for laboratory and field strains of S. granarius and T. castaneum. The laboratory strains (SgLab and TcLab) originated from non-resistant strains maintained at Crop Research. Institute. The field strains were obtained from the resistance screening: the most resistant strains of each tested species (S. granarius; T. castaneum) from the previous screening were selected. The field bioassays of adults and eggs of S. granarius (SgPod) and T. castaneum (TcEt) were performed using plastic containers (volume 125 ml; 52 mm in diameter, 74 mm in height; Gosselin SAS, France) with lids and bottoms covered by Miralontextile mesh (UHELON, polyamid, 9.5139; Silk and Progress s.r.o., Czech Republic) to enable gas penetration. At each of the three locations, a set of 3 bioassay containers was placed that included 50 adults and commodity with pest eggs. To obtain commodity with
eggs, 50 unsexed adults (we assumed a 50% sex-ratio) were placed in containers and left to oviposit (at 25e26 C and 75% RH) for 72 h before fumigation. For each species, different commodities were used: wheat 100 ± 1 g for S. granarius and 80 ± 1 g wheat and 10 ± 1 g oat flakes for T. castaneum. Adults were gently removed shortly before transportation of the vials to the fumigation site from the laboratory. Before sheeting the stacks, vials with biological samples of laboratory sensitive strains and field resistant strains were placed at three positions (Fig. 1): 2 positions under the sheets and 1 position outside the sheets. Each position received 3 replicates from each tested species, strain and stage (i.e., adults or eggs). Efficacy evaluation and statistics. Adult mortality was evaluated immediately after the termination of treatment. The fumigation efficacy on eggs was further monitored at 55 days after the end of exposure for both species. The results were statistically
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fumigated parts of the store outside the tarpaulin indicated poor sealing, likely due to the very thin PVC tarpaulin used. Table 3 summarizes the information on the mortality of adults of the laboratory and field strains of T. castaneum (TcEt) and of S. granarius (SgPod) at the three positions and the control after the fumigation period. The effects of strain and position were significant (ANOVA; F/9,32/¼ 2.935, p ¼ 0.01). As shown in Table 3, even low-dose fumigation (Fig. 1A) resulted in 100% adult mortality of both laboratory and field strains of S. granarius and laboratory strains of T. castaneum. However, the mortality of the field (i.e., resistant) strain of T. castaneum ranged from 47% to 95%. Table 4 presents larval emergence - expressed as number of larvae emerged per container for the field and laboratory strains of T. castaneum and S. granarius at the three positions and the control after the fumigation period. The effects of strain and position were significant (ANOVA; F/9,32/¼ 8.88, p<0.001). Larval emergence from the fumigated commodity samples with pest eggs was zero for T. castaneum or near zero for S. granarius in laboratory strains. Different situations were observed for the field strains of both species, where 1.3e6.0 emerged larvae per 100 g of phosphinefumigated grain for S. granarius and 63.7e80.0 emerged larvae per 100 g of fumigated cereal flakes for T. castaneum. 4. Discussion Fig. 1. Schematic picture of the flat store with treated commodity and the three positions of the bioassays where the phosphine concentration was measured.
processed and evaluated with the statistical program Statistica 12 using two-way ANOVA. Adult mortality and fumigation efficacy on eggs were separated by post hoc Tukey’s HSD test. Before analysis, all data were submitted to normalization of variance (ln (xþ1)). Means (±SE) of untransformed data are reported. 3. Results 3.1. Survey in stores, strain collection and resistance screening Of the 86 grain stores sampled, 27 stores were positive for any level of T. castaneum, S. granarius or both. We collected 23 strains of S. granarius and 8 strains of T. castaneum for resistance testing. The results of the logistic regression knock-down model and ANOVA evaluation of KT100 (based on DDPRK protocol) are summarized in Tables 1 and 2 Significant differences in the KT100 values were noted in the knock-down time between the tested strains for both species, as shown in Table 1 for S. granarius (F ¼ 28.4; df ¼ 23; p < 0.001) and in Table 2 for T. castaneum (F ¼ 721.6; df ¼ 8; p < 0.001). In the pooled data for both species of stored product pest beetles, we found that of all (n ¼ 31) tested field strains, 38.7% (evaluation by KT100) had at least a minimum level of resistance with a coefficient of 1.1 or higher. In the tested strains, the resistance coefficient KT100 ranged from 0.5 to 2.3 for S. granarius (Table 1) and from 0.9 to 52.5 for T. castaneum (Table 2). 3.2. Field validation of fumigation efficacy in field and laboratory strains Fig. 2 shows the course of concentrations, and Tables 3 and 4 show a summary of the Ct products obtained for each measured position (Fig. 1.). The calculated Ct ranged from 5.9 to 7.4 g*h/m3. The maximum concentration achieved under the tarpaulin was 115 ppm after 24 h from the phosphide tablet introduction. The low phosphine concentration reached under the tarpaulin and the relatively high phosphine concentration detected in the non-
This paper presents analyses of the data from the first study in the Middle European ecoregion concerning the differential sensitivity to phosphine insecticide of various strains of two species of stored-product Coleoptera pests collected during a 3-year grain store survey. For the first historical screening of phosphine resistance in Czech stores, the standard knock-down “Detia Degesch Phosphine Resistance Test Kit (DDPRK) (Steuerwald et al., 2006) was used. DDPRK test belongs to a group of so-called quick resistance tests (Reichmuth, 1991; Chen et al., 2015). As the resistance may be determined in less than one day, these approaches shorten the time needed to assay for resistance in comparisons with a standard FAO phosphine resistance test (FAO, 1975). In our work we decided not to employ the original method (Reichmuth, 1991) but the DDPRK version since it is commercially available and therefore the achieved results can be compared with other studies based on this commercial kit (e.g. Agrafioti et al., 2017). However, the results obtained by the DDPRK quick tests are to some extent compatible even with different methodical approaches. Several studies have shown that the knock-down based kit generates results comparable to those of the more labour- and time-demanding FAO-method (FAO, 1975). For example, Cao et al. (1998) documented a strong positive linear correlation between the logLC50 values (obtained by the FAO method) and logKT50 values obtained by the quick Reichmuth (1991) knock-down method on which the DDPRK is based. Recently, the FAO methods (FAO, 1975) and commercial DDPRK were validated by Agrafioti et al. (2017), and similar results regarding the compatibility of both tests were confirmed. Nevertheless, our study is the first to show that even for a single experimental testing procedure (i.e., DDPRK) using various types of coefficients of resistance, the evaluation of identical data may yield some differences. For example, the comparison of S. granarius strains by KT100 (i.e., calculated according to the instructions provided by the DDPRK) showed only 21.7% of strains with increased resistance, but the KT99 (calculated by the standard logistic regression knock-down model) showed a value of 82.6%. We therefore included both coefficients in this study. Nevertheless, irrespective of data interpretation differences, our results consistently demonstrated that although S. granarius was more
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111
Concentra on (ppm)
140 120 100 80 60 40 20 0 0
24
47.5
72.5
95.5
Exposure me (hours) Posi on no. 1
Posi on no. 2
Posi on no. 3
Fig. 2. Phosphine concentration reached at the three positions (under and outside the sheets) during the course (95,50 h) of field commercial fumigation.
Table 3 Percentage mortality (mean ± SE) per container of adults of laboratory and field strains of Tribolium castaneum and of Sitophilus granarius at the three positions and the control after the fumigation period. Tribolium castaneum
Position no. 1 Position no. 2 Position no. 3 Control
CT - product (g*h/m3)
Sitophilus granarius
Laboratory strain
Field strain
Laboratory strain
Field strain
100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 1.3 ± 0.7a
18.7 ± 2.4b 95.3 ± 2.7c 46.7 ± 5.7bc 1.3 ± 1.3a
100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 2.0 ± 1.2a
100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 2.7 ± 0.7a
3.3 5.9 7.4 0.0
The means followed from the same lowercase letter are not significantly different; HSD test at 0.05.
Table 4 Adult emergence (mean ± SE) per container for the field and laboratory strains of Tribolium castaneum and of Sitophilus granarius at three positions and the control after the fumigation period. Tribolium castaneum
Position no. 1 Position no. 2 Position no. 3 Control
CT - product (g*h/m3)
Sitophilus granarius
Laboratory strain
Field strain
Laboratory strain
Field strain
0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 134.0 ± 44.1d
85.7 ± 29.2cd 63.7 ± 14.6cd 80.0 ± 45.5cd 98.0 ± 48.2cd
4.7 ± 1.3 ab 0.0 ± 0.0a 0.3 ± 0.3a 90.7 ± 12.0cd
17.3 ± 12.4bc 1.3 ± 0.7 ab 6.0 ± 4.0 ab 81.0 ± 20.1cd
3.3 5.9 7.4 0.0
The means followed from the same lowercase letter are not significantly different; HSD test at 0.05.
frequently found in stores than was T. castaneum, its level of phosphine resistance was neither very frequent nor very high. However, a higher level of phosphine resistance was found in several strains of T. castaneum. For example, one of the tested T. castaneum strains was moving after 420 min of continual phosphine exposure; thus, this strain was approximately 52.5 more tolerant than the sensitive laboratory strain, for which movement stops in less than 8 min. This result can be considered a relatively high resistance level since Reichmuth (1991) described resistant insects as those that are still moving after 30 min of exposure to phosphine at 1 ml l1. Monro and Bond (1972) demonstrated that it took more than 28 generations to obtain a phosphine resistance factor exceeding 3 in S. granarius under laboratory conditions. Although it may seem that this species is not prone to quickly acquire phosphine resistance, there is little evidence for this idea under field grain store
conditions. For S. granarius, there are only two published reports on phosphine field resistance, and these reports provide contradictory results. While Ignatowicz (2000) found this species resistant, Kocak et al. (2018) reported that among the tested species and strains collected from Turkey grain stores, S. granarius was far less resistant to phosphine than were other species; e.g., the maximal coefficient of resistance was only 5.4 for S. granarius, while it was 200.5 for S. oryzae and 459.6 for O. surinamensis. Our results seem to stand between those of Kocak et al., (2018) and Ignatowicz (2000), showing a moderately decreased level of phosphine sensitivity in two strains of S. granarius, while the remaining tested strains were sensitive or very sensitive. Although results from more countries are needed to describe the extent or geographical distribution of the resistance of S. granarius, a different situation can be found for T. castaneum, the other species tested in our study. Our results on the sensitivity of T. castaneum to phosphine seem to fit within the
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broadly reported resistance trends from many geographical ecoregions, showing that the frequency of T. castaneum phosphine resistance in strains is increasing and becoming a global issue (e.g., Australia - Daglish et al., 2015; Nayak et al., 2017; North America e Opit et al., 2012; Asia - Koçak et al., 2018). It is important to note that quick knock-down resistance tests are mainly useful for an initial resistance surveys, such as our study. Although they can be effectively used to detect resistance (i.e. presence/absence) they do not serve well to thoroughly measure the strength of resistance. Particularly, they are not currently designed to accurately distinguish between a weak and strong resistance type (Chen et al., 2015). This may be a major drawback of these methods since distinguishing weak from strong resistance may have profound impact on evaluation of fumigation protocols. For example, the published work from USA (Opit et al., 2012) indicates, that it is likely that weakly resistant populations can be controlled with recommended fumigations that hold 200 ppm for at least three days (Phillips et al., 2012), whereas the strongly resistant populations cannot. Currently there is no published information on strong resistance concerning S. granarius. In contrast to previous species, there is a convincing evidence for occurrence of strong resistance to T. castaneum in several geographical regions (Opit et al., 2012; Koçak et al., 2015; and Nayak et al., 2017). Regarding our work, it may by hypothesised - though not explicitly tested with the method used-that at least one of the T. castaneum strains may be strongly resistant to phosphine, while the others are more likely to be weakly resistant. However, this hypothesis has to be verified by the molecular methods (e. g. Chen et al., 2015). As mentioned in the introduction, resistance to phosphine is a serious issue in several geographical areas from which it may spread around the world. From an international perspective, our resistance survey can be considered a useful missing piece of information from the Middle European region that helps to complete the global mosaic picture showing the current geographical phosphine resistance distribution. However, the resistance survey is only the first part of resistance management (Nayak et al., 2017). Agricultural and food industry practice is interested in not only whether resistance is present in a given area but also if and how the current local strength of resistance to phosphine can project into fumigation protocols (i.e. optimization of phosphine dosage and exposure - Meyvaci et al., 2010) and if there is a need for any label adjustments. We therefore compared the efficacy of phosphine laboratory strains under conditions of commercial fumigation. Our concentration measurements showed that for the observed fumigation, the fumigation company used improper plastic sheeting and attachment to the floor, leading to relatively high phosphine concentrations in the store but low phosphine concentrations and Ct products under the sheets. Wang et al. (2006) documented that the type of sheeting material and its thickness and quality of attachment to the floor strongly affect the retention of fumigant under the tarpaulin. We found similar results for the Czech populations. Even very low concentrations and Ct products, reached under the sheets due to poor sealing, were able to control non-resistant adults and egg stadia in both tested species. We recorded zero survival of lowresistance field strains of S. granarius. The high mortality at low concentrations was likely due to high temperatures (25 C) occurring during fumigation. Previously, it was shown in Czech mill fumigation that high temperature may play a significant role in compensation for a lower phosphine dosage, while the highest concentration achieved at the lowest temperature resulted in egg survival (Aulicky et al., 2015a). In our study, we observed high survival (approximately 50%) of substantially more resistant strains of T. castaneum and emergence from eggs. Our experiment thus confirmed the findings of Wang et al. (2006), who showed that the quality of sealing was a more important factor in controlling
Southern China resistant than sensitive strains of storage pests. Although a single fumigation event, executed under poorly sealed conditions, usually results in little population resistance shift, multiple fumigations of leaky structures is the most likely factor that triggers development of stored product pest resistance to phosphine (Collins, 2009). This indicates that the problem of improper sealing not only hampers phosphine efficacy on resistant strains but may even be a cause of resistance selection and occurrence. For example, a recent Australian analysis by Nayak et al. (2017) showed that resistant populations are especially reported from stores showing improper levels of sealing. It was also found that strong resistance development in T. castaneum was more prevalent in unsealed bunkers, a trend that was also previously reported for S. oryzae (Holloway et al., 2016) and R. dominica (Collins et al., 2017). Their hypothesis is that generally in poorly sealed structures, it is difficult to achieve and maintain not only proper concentration but also gas distribution and commodity penetration. As a result, repeated fumigations to overcome residual populations are not uncommon in structures in which patterns are associated with resistance selection. Our results, along with several other studies (Aulicky and Stejskal, (2015b), Collins et al., 2017; Nayak et al., 2015, 2017), imply that the outcome of phosphine fumigation in the field is a relatively complex interplay between three groups of factors: environmental factors (temperature, wind, and humidity), quality of fumigation technological procedure (sealing, proper dosage, even gas distribution, etc.) (Bell, 2014) and level of pest resistance. In commercial phosphine fumigation, various factors can either enforce (compensate) or weaken other factors. It seems that, for example, excellent sealing and high temperature may partially compensate for the negative effects of low gas dosage or the presence of pest resistance. Conclusions and practical implications. The threat to the future use of phosphine fumigants is not only the frequency of resistance but also its potential increase in strength. Our study demonstrates that, although a high proportion of the tested Czech pest populations were still sensitive, several populations (notably T. castaneum) showed alarming levels of phosphine resistance. Such elevated level of resistance may have impact on the practical use of phosphine in Czech Republic. For example, a decreased field phosphine fumigation efficacy on certain populations of T. castaneum may be encountered, especially under suboptimal dosage conditions. The currently used phosphine EU labels include condition-dependent range of doses and exposures. In case that resistant population occurs, the upper marginal values, from the legally registered range of doses and exposures, should be used; and vice versa. Therefore, farms, mills and commodity traders should apply routine resistance screenings before any fumigation and modify fumigation protocol accordingly. The particular fumigation protocol should also take into account a potential occurrence of weak and strong population resistance to phosphine. In future, it even may be required to alter phosphine rates and duration in order to control the strong resistant populations. However, the practice is currently challenging a problem how to quickly and cheaply determine strong resistance. The molecular methods are costly and the currently used knock-down quick resistance tests are not able to distinguish a weak from a strong resistance. It implies that further research in that venue is still urgently needed. Not only the consequences but also the causes of resistance should be addressed. Pest resistance to fumigants has mainly been associated with high fumigation frequencies and improper fumigation practices (Bell, 2014). The long-term battle against resistance should therefore be based on the support and implementation of anti-resistance strategies that should include fumigation training, extension, the penalization of improper application methods and fumigation malpractice (e.g., Aulicky et al., 2015) and the
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introduction of alternative control methods and active ingredients (e.g., Nayak et al., 2016) with the potential to break the selection process for phosphine resistance. Acknowledgements We thank Sarka Tuckova for excellent technical assistance. This study was supported by the Ministry of Agriculture of the Czech Republic, Projects No. RO 0419 and QJ1530373. References €tze, C., Allegra, J., Athanassiou, C.G., 2017. Evaluation Agrafioti, P., Sotiroudas, V., Go of phosphine resistance in stored-product insects from Greece using two assessment methods. In: Trematerra, P., Trnad, S. (Eds.), Proc. of the 11th Int. Conf. on the IOBC-WPRS Bulletin. Ljubljana, vol. 130, p. 95. Aulicky, R., Stejskal, V., Frydova, B., Athanassiou, C.G., 2015. Susceptibility of two strains of the confused flour beetle (Coleoptera: Tenebrionidae) following phosphine structural mill fumigation: effects of concentration, temperature, and flour deposits. J. Econ. Entomol. 108, 2823e2830. Aulicky, R., Stejskal, V., 2015. Efficacy and limitations of phosphine “spot-fumigation” against five Coleoptera species of stored product pests in wheat in a grain store. Plant Protect. Sci. 51, 33e38. Bell, C.H., 2014. A review of insect responses to various encountered in the managed storage environment. J. Stored Prod. Res. 59, 260e274. Cao, Y., Zhang, J.J., Bengston, M., 1998. Studies on a quick method to measure resistance of four strains of Tribolium castaneum (Herbst) to phosphine. In: Jin, Z.X., et al. (Eds.), Stored Product Protection. Proc. Of the 7th Int. Conf. on Stored-Product Protection, PR China, pp. 603e606. Champ, B.R., Dyte, C.E., 1976. Report of the FAO global survey of pesticide susceptibility of stored grain pests. In: FAO Plant Prot. And Prod. Series, No. 5. FAO, Rome. Chen, Z., Schlipalius, D., Opit, G., Subramanyam, B., Phillips, T.W., 2015. Diagnostic molecular markers for phosphine resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica. PLoS One 10 e0121343. Collins, P.J., 2009. Strategy to Manage Resistance to Phosphine in the Australian Grain Industry. An Initiative of the National Working Party on Grain Protection. Cooperative Research Centre for National Plant Biosecurity Project. CRC70096. http://www.graintrade.org.au/sites/default/files/file/Phosphine_Strategy.pdf. Collins, P.J., Falk, M.G., Nayak, M.K., Emery, R.N., Holloway, J.C., 2017. Monitoring resistance to phosphine in the lesser grain borer, Rhyzopertha dominica, in Australia: a national analysis of trends, storage types and geography in relation to resistance detections. J. Stored Prod. Res. 70, 25e36. Daglish, G.J., Nayak, M.K., Pavic, H., Smith, L.W., 2015. Prevalence and potential fitness cost of weak phosphine resistance in Tribolium castaneum (Herbst) in Eastern Australia. J. Stored Prod. Res. 61, 54e58. FAO, 1975. Method No. 16. Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides. Tentative method for adults of some major pest species of stored cereals, with methyl bromide and phosphine. Plant Protect. Bull. FAO. 23, 12e25. Holloway, J.C., Falk, M.G., Emery, R.N., Collins, P.J., Nayak, M.K., 2016. Resistance to phosphine in Sitophilus oryzae in Australia: a national analysis of trends and frequencies over time and geographic spread. J. Stored Prod. Res. 69, 129e137.
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Ignatowicz, S., 2000. Cases of phosphine resistance in the grain weevil, Sitophilus granarius, found in Poland. In: Zuxun, J., et al. (Eds.), Proc. 7th Int. Working Conf. on Stored-product Protection, Beijing, vol. 1. Sichuan Publishing House of Science & Technology, Chengdu, China, pp. 625e630. Koçak, E., Schlipalius, D., Ramandeep, K., Tuck, A., Ebert, P., Collins, P., Yilmaz, A., 2015. Determining phosphine resistance in rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) populations from Turkey. Turkiye Entomoloji Dergisi 39, 129e136. Koçak, E., Yilmaz, A., Alpkent, Y.N., Ertürk, S., 2018. Phosphine resistance to some coleopteran pests in stored grains across Turkey. In: Trematerra, P., Trdan, S. (Eds.), Proc. of the 11th Int. Conf. on the IOBC-WPRS Bulletin. Ljubljana, vol. 130, pp. 303e310. Meyvaci, K.B., Sen, F., Aksoy, U., 2010. Optimization of magnesium phosphide treatment used to control major dried fig storage pests. Horticult. Environ. Biotechnol. 51, 33e38. Monro, H.A.U., Bond, E.J., 1972. Resistance of a laboratory strain of Sitophilus granarius (rkL) (Coleoptera, Curculionidae) to phosphine. J. Stored Prod. Res. 8, 199e207. Nayak, M.K., Daglish, G.J., Phillips, T.W., 2015. Managing resistance to chemical treatments in stored products pests. Stewart Postharvest Rev 11, 1e6. Nayak, M.K., Jagadeesan, R., Kaur, R., Daglish, G.J., Reid, R., Pavic, H., Smith, L.W., Collins, P.J., 2016. Use of sulfuryl fluoride in the management of strongly phosphine-resistant insect pest populations in bulk grain storages in Australia. J. Grain Storage Res. 16, 100e107. Nayak, M.K., Falk, M.G., Emery, R.N., Collins, P.J., Holloway, J.C., 2017. An analysis of trends, frequencies and factors influencing the development of resistance to phosphine in the red flour beetle Tribolium castaneum (Herbst) in Australia. J. Stored Prod. Res. 72, 35e48. Opit, G.P., Phillips, T.W., Aikins, M.J., Hasan, M.M., 2012. Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma. J. Econ. Entomol. 105, 1107e1114. Phillips, T., Thoms, E., Demark, J., Walse, 2012. Fumigation, chapter 14. In: Stored Product Protection, vols. 156e4. K-State Research and Extension publication, pp. 1e14. Reichmuth, C.A., 1991. Quick test to determine phosphine resistance in stored products research. GASGA Newsletter 15, 14e15. Stejskal, V., Hubert, J., Kucerova, Z., Munzbergova, Z., Lukas, J., Zdarkova, E., 2003. The influence of the type of storage on pest infestation of stored grain in the Czech Republic. Plant Soil Environ. 49, 55e62. Stejskal, V., Aulicky, R., Kucerova, Z., 2014. Pest control strategies and damage potential of seed-infesting pests in the Czech stores e a review. Plant Protect. Sci. 50, 165e173. Stejskal, V., Hubert, J., Aulicky, R., Kucerova, Z., 2015. Overview of present and past and pest-associated risks in stored food and feed products: European perspective. J. Stored Prod. Res. 64, 122e132. Steuerwald, R., Dierks-Lange, H., Schmitt, S., 2006. Rapid bioassay for determining the phosphine tolerance. In: Proc. Of the 9th Int. Working Conf. on StoredProduct Prot., 15-18 October 2006, Campinas, Sao Paolo, BRAPOS, Passo Fundo, RS, Brazil, pp. 306e311. Taylor, R.W.D., Halliday, D., 1986. The geographical spread of resistance to phosphine by Coleopterous pests of stored products. In: Proceedings British Crop Protection Conference, Pests and Diseases, pp. 607e613. Brighton. Wang, D., Collins, P.J., Gao, X., 2006. Optimising indoor phosphine fumigation of paddy rice bag-stacks under sheeting for control of resistant insects. J. Stored Prod. Res. 42, 207e217.