Phosphine is the primary fumigant to disinfest majority of world's grain against insect pests. However, the toxicity of phosphine against stored grain insect populations from different locations in India has been compared rarely. Under the present study, comparative toxicity of phosphine was determined for the storage insect pests of wheat and rice at different locations. It also evaluated phosphine’s efficacy against Callosobruchus maculatus (Fabricius) and Hypothenemus hampei (Ferrari) for which information is limited. Developmental stages including adults, larvae and eggs of red flour beetle, Tribolium castaneum (Herbst), adults of rice weevil, Sitophilus oryzae (L.) and lesser grain borer, Rhyzopertha dominica (F.), larvae of khapra beetle, Trogoderma granarium Everts, eggs of C. maculatus and larvae, pupae and adults of H. hampei, were exposed to phosphine concentrations ranging from 0.001 to 2.25 mg/L for 24, 48, and 72 hours. Sitophilus oryzae and R. dominica strains of Almora were observed to be more resistant to phosphine when compared with the most susceptible population from Cuttack. Tribolium castaneum strains of Delhi and Almora were more susceptible than strains of Cuttack. All the tested developmental stages of H. hampei were found to be highly susceptible; while, eggs of C. maculatus and larvae of T. granarium were highly resistant. Results showed significant variations in susceptibility among insect populations and life stages. The study also revealed that median lethal concentrations decrease with an increase in exposure levels irrespective of the insect species and life stages tested, suggesting the need for location-specific dosage and exposure periods for phosphine fumigation.
Research Article
Unmasking phosphine: Assessing its continued effectiveness on stored product insects in India across populations and life stages
https://doi.org/10.21203/rs.3.rs-4206423/v1
This work is licensed under a CC BY 4.0 License
You are reading this latest preprint version
phosphine toxicity
stored grain insects
cereals
pulses
coffee berry
Food products stored for a few months or for a longer period get infested by insect pests leading to economic losses, and generally require fumigation for disinfestation (Hagstrum, Phillips & Cuperus, 2012). The post-harvest losses in stored food grains account for about 20–30% in tropical countries and 5–10% in temperate regions (Nakakita, 1998; Rajashekar, Gunasekaran & Shivanandappa, 2010). Food and Agriculture Organization of the United Nations has reported the global post-harvest losses of food grains amounting approximately to 1.3 billion tonnes annually (Gustavsson, Cederberg, Sonesson, van Otterdijk & Meybeck, 2011). Among storage insects, red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae); rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae); lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) (Arora, Stanley & Srivastava, 2021); and pulse beetle, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) (Arora & Srivastava, 2021) are the most common ones which cause extensive damage to the stored grains and pulses. The khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), is an extremely dangerous invasive pest of stored products of plant and animal origin (Kavallieratos, Athanassiou, Boukouvala & Tsekos, 2019). It exists in Asia, Europe, and Africa, but has also been intercepted in the USA and Australia. Currently, it is categorized as a quarantine pest in Belarus, Canada, Mexico, Morocco, New Zealand, the USA, and other countries (EPPO, 2022; Myers & Hagstrum, 2012). Furthermore, Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) is the most serious pest of stored coffee beans causing worldwide losses of over US$500 million, considering that there are 82 coffee-producing countries worldwide (FAOSTAT, 2019). The infestation of coffee berries could be > 90% at harvest leading to reduced yield and quality of the final product (Adriano et al. 2021), resulting in considerable economic losses (Angelica et al. 2019; Peter, 2018). No reports are available for a simple and cheap method of controlling H. hampei (CABI, 2019).
Phosphine has been used worldwide as an ideal fumigant for several decades for protecting stored products against insect pests (Nayak, Daglish, Phillips & Ebert, 2020). The phasing out of methyl bromide, an ozone-depleting substance, honouring the Montreal Protocol, raises phosphine’s importance as a fumigant. Several countries are developing phosphine fumigation protocols as a substitute for methyl bromide for its application in quarantine and pre-shipment treatment of fresh agricultural produce (fruits and vegetables, cut flowers), dried fruits, timber, coffee, and cocoa beans (Kumar, 2018). In the present scenario, there is hardly any alternative to phosphine, mostly due to its low price, practicality, proven effectiveness against a broad spectrum of pests, compatibility with most storage conditions, and international acceptance as a residue-free treatment.
Phosphine has the intrinsic disadvantage of permitting insects subjected to sub-lethal concentrations to become resistant to the fumigant swiftly and laboratory selections have proved the same (Winks, 1986). Lack of airtightness conditions in storage units is likely the main reason of sub-lethal exposure. If these sub-lethal exposures are repeated frequently, which is common in many areas of the world, selection of phosphine-resistant individuals is possible (Chaudhry, 1997). Sitophilus granarius (L.) is the first storage insect that was found to have exhibited phosphine resistance. Monro, Musgrave & Upitis (1961) first observed that after selection with the fumigant methyl bromide, a strain of S. granarius developed phosphine resistance as cross-resistance. Reports of a survey conducted by Food and Agriculture Organization (FAO) globally during 1972–1973 revealed that 10 percent of storage pest populations sampled were found to be resistant to phosphine (Champ & Dyte, 1976). Despite being an ideal fumigant, control failures have been reported in various countries in the last 40 years after this global survey which suggested that higher levels of resistance to phosphine have been developed in some of the storage insect pests (Chen, Schlipalius, Opit, Subramanyam & Phillips, 2015; Collins, Daglish, Bengston, Lambkin & Pavic, 2002; Gautam, Opit, Konemann, Shakya & Hosoda, 2020; Kaur et al. 2015; Ling, 1999; Lorini, Collins, Daglish, Nayak & Pavic, 2007; Nayak et al. 2013, 2020; Opit, Phillips, Aikins & Hasan, 2012; Rajendran, 1999; Ramya, Srivastava & Subramanian, 2018; Ramya, Srivastava, Subramanian & Ranjith, 2023). In North America, surveys in the state of Oklahoma showed a significant increase in the frequency of resistance in both T. castaneum and R. dominica over two decades (Opit et al. 2012). Subsequent work in North America highlighted increases in the frequency and strength of phosphine resistance in common stored product pests like R. dominica (Afful, Brent, Nayak & Thomas, 2018) and T. castaneum (Cato, Elliott, Nayak & Phillips, 2017; Gautam, Opit and Hosoda, 2016). Resistance frequencies for some populations of these species were in the higher range of 80–100%. Reports of phosphine resistance in South America include that of Lorini et al. (2007), who surveyed R. dominica populations in Brazil and diagnosed 14 of the 19 samples as strongly resistant. Research in countries in Asia reported strongly resistant populations of R. dominica from Bangladesh (Mills, 1983), India (Rajendran, 1999), China (Ren, O’Brien & Whittle, 1994), and The Philippines (Acda, 2000); S. oryzae from India (Rajendran, 1999), Vietnam (Nguyen, Collins, Duong, Schlipalius & Ebert, 2016), and China (Ling, 1999); Cryptolestes ferrugineus (Stephens) from China (Ling, 1999); Liposcelis entomophila (Enderlein) from Indonesia (Pike, 1994) and China (Cao, Son and Sun, 2003); L. bostrychophila from India (Rajendran, 1994); and T. granarium from Pakistan (Athanassiou, Phillips & Wakil, 2019).
Under the present study, the relative susceptibility of populations of R. dominica, S. oryzae, T. castaneum and T. granarium from different locations to phosphine was determined to assess the locational impact, if any on their susceptibility to phosphine. In addition to these, the efficacy of phosphine was also tested against the life stages of two more storage insects, i.e., C. maculatus and H. hampei as information available for these is scanty.
Laboratory bio-assay experiments were conducted with field-collected populations of R. dominica, S. oryzae, T. castaneum and T. granarium from the Food Corporation of India (FCI) godowns of Delhi, Almora, and Cuttack (Fig. 1) to see the variability, if any, in the susceptibility among different populations and also among the different developmental stages of the storage pests to phosphine. The field populations were collected and maintained in the laboratory. The F1 generation was used to conduct the bioassays. The intention was to see the differential susceptibility levels of different populations of four storage insect pest species since the susceptibility of a particular population depends on the previous exposure to the fumigant and the conditions of fumigation in the storage godowns. Studies were also conducted to assess the efficacy of phosphine against the egg stage of C. maculatus and the different life stages of H. hampei. The ages of different life stages of test insects undertaken for experiments were 4–5 days old adults, 3rd instar larvae, 2–3 days old eggs of T. castaneum; 4–5 days old adults of S. oryzae and R. dominica; 3rd instar (15–20 days old) larvae of T. granarium; 1 day old eggs of C. maculatus, 2 weeks old adults, 1 week old larvae and 2 days old pupae of H. hampei.
2.1. Rearing of test insects
The field-collected insect populations of T. castaneum were reared in the laboratory by maintaining the culture at 30 ± 20C and 70% relative humidity (RH) on sterilized wheat flour mixed with 5% Brewer’s yeast following the methodology of Bhatia & Pradhan (1968). The immature egg stages were separated using sieve no. 100 (150 microns) (unique standard test sieves as per ISS: 460) (manufactured by Jayant Test Sieves). The field populations of S. oryzae and R. dominica were maintained on rice grains at 28 ± 20C and 65 ± 5% R.H. following the methodology mentioned by Miller, Phillips & Cline, 1969. The different field-collected populations of T. granarium were reared on crushed wheat grains at 35-400C and 65 ± 5% RH in the incubator as per the methodology by Yadav, Srivastava & Subramanian, 2020.
In addition to the comparison of susceptibility levels in different populations of T. castaneum, S. oryzae, R. dominica and T. granarium, we also intended to study the efficacy of phosphine against C. maculatus and life stages of H. hampei. Hence, the laboratory-reared cultures of both these species were maintained for bioassay studies. The nucleus culture of C. maculatus was procured from the Division of Entomology, IARI, New Delhi, and reared on green gram grains at 27 ± 30C and 70–75% relative humidity following the methodology of Arora and Srivastava (2021). The insect population of H. hampei was collected from the coffee gardens of Central Coffee Research Station, Chikmagalur, Karnataka, India, and Regional Coffee Research Station, Wayanad, Kerala, India. The infested ripe berries were collected from the fields and brought to the laboratory for maintaining culture. The population was reared and maintained under controlled conditions at 24-260C and 70–75% relative humidity on fresh ripe coffee berries.
2.2. Phosphine generation and bioassay technique
Phosphine gas was generated following the FAO (1975) method. Commercially available Aluminium phosphide tablets (Celphos − 56% Aluminium phosphide and 44% Ammonium carbamate) (UPL Ltd. India) were used for generating phosphine gas by submerging the tablet in 5% sulphuric acid underneath a collection tube (local manufacturer at Delhi, India). The collection tube was specially fabricated with a wide mouth at one end and a narrow mouth at the other end which can be tightly closed using a silicon septum. The apparatus for gas generation contained a glass container and collection tube filled with 5% sulphuric acid, with the top of the collection tube submerged below the surface of the liquid to displace the air. Once all the air was displaced by the liquid in the collection tube, a silicon septum was fitted to the top of the collection tube whilst still submerged. A tablet containing aluminium phosphide was wrapped in a muslin cloth and dropped into the glass jar. A glass funnel was then placed over the top of the tablet and the weight of the funnel carried the tablet to the bottom of the glass container. The collection tube was then manoeuvred over the funnel opening and clamped in place. A small positive pressure was created on the surface of the collected gas by keeping the level of the liquid in the glass container higher than that of the collecting tube. Hamilton gas-tight syringe was used to draw the gas from the generated source and injected into the desiccator (local manufacturer in Delhi, India) for bioassay.
The phosphine bio-assay experiments were conducted in glass desiccators (volume ~ 2.5 L) annexed with inlet and outlet tubes for gas monitoring, besides a tube in the center for gas injection through septa. The plastic boxes (7 cm × 4 cm) containing each life stage of insects separately along with food were placed inside the desiccators, and dosed with known concentrations of phosphine. Phosphine concentration was monitored initially using phosphine gas monitor FumiSensePro (UPL Pvt Ltd., 1-2000 ppm) when gas was injected in the desiccators, and towards the end of the exposure period. The volume of each desiccator was measured by the water displacement method. The volumes of phosphine to be injected were calculated using the source concentration, required dose, and volume of the desiccators as follows:
Where, D1 – volume of phosphine gas to be injected
X1 – desired concentration in a desiccator
V1 – volume of desiccator
X2 – concentration of Phosphine source (1200 ppm) (mgL− 1)
Test insects for laboratory bioassay included 50 each of the different life stages of T. castaneum (adults, larvae, and eggs), S. oryzae (adults), R. dominica (adults), T. granarium (larvae), C. maculatus (eggs) and H. hampei (adults, larvae and pupae) placed in airtight desiccators. The experiments were conducted in five replicates using 7 to 8 phosphine concentrations (0.25–3.0 mg/L) including control, to establish each concentration-mortality curve. However, the minimum dosage of 0.25 mg/L was further lowered to 0.01 to 0.001 mg/L for evaluating LC50 values of fumigant due to higher insect mortality (60–70% for S. oryzae and R. dominica and 90% for coffee berry borers). The gas concentrations tested against different life stages of H. hampei strains were in the range of 0.001 to 0.035 mg/L. The bio-assay experiments were conducted to assess the variation in susceptibilities of life stages of T. castaneum, S. oryzae, R. dominica, and T. granarium at three locations, Delhi, Cuttack, and Almora for three exposure periods, i.e., 24 hours, 48 hours and 72 hours. Experiments were also carried out to evaluate the efficacy of phosphine against the egg stage of C. maculatus and different life stages (larva, pupa, and adult) of H. hampei for three exposure periods. After the exposure period, a small amount of the culture media was added to each insect vial before they were transferred to a controlled environment of 24-260C and 55–60% relative humidity where they were held until mortality was assessed after 7 days. In the case of H. hampei, a small quantity of coffee berry borer diet (Brun, Gaudichon & Wigley, 1993) was added to the vials before transferring them to the control conditions.
2.3. Statistical analysis
The mortality data was subjected to Abbott’s correction (Abbott, 1925) and then probit Analysis using EPA Probit Analysis Program Ver. 1.5. Resistance ratio (RR) was computed from dose-mortality data subjected to probit analysis for a given population for which the LC50 of the population was divided by the LC50 of the susceptible population. Since we did not have an established susceptible population of test insects, we calculated RR50 by comparing LC50 of populations of test insect strains with that of the least resistant population. Hence, RR was used as a measure of variation in resistance between field populations. RR50 values were calculated for all the tested life stages of T. castaneum, S. oryzae, R. dominica, and T. granarium at different locations.
The comparative susceptibility of different life stages tested and the different populations are explained as below. The probit analysis of mortality data of storage insects of cereals, pulses, and coffee beans is presented in Table 1–5. The RR50 values evaluated for the life stages of each test insect strain of different locations are also presented in Table 1–4.
| Location | Exposure period (hrs) | LC50 (95% Fiducial limit†) (mg/L) | LC95 (95% Fiducial limit†) (mg/L) | Resistance Ratio‡ | Slope ± SE | χ2 calc. |
|---|---|---|---|---|---|---|
| Delhi | 24 | 0.303 (0.16–0.572) | 5.420 (2.860–10.240) | 75.75 | 1.343 ± 0.141 | 0.875 |
| 48 | 0.093 (0.035–0.247) | 6.160 (2.330–16.310) | 93.00 | 0.917 ± 0.216 | 0.920 | |
| 72 | 0.068 (0.028–0.165) | 2.940 (1.210–7.160) | 68.00 | 1.045 ± 0.197 | 0.958 | |
| Cuttack | 24 | 0.004 (0.002–0.005) | 0.058 (0.038–0.103) | 1 | 1.391 ± 0.155 | 10.121 |
| 48 | 0.001 (0.001–0.002) | 0.029 (0.018–0.056) | 1 | 1.237 ± 0.159 | 4.911 | |
| 72 | 0.001 (0.0007–0.001) | 0.021 (0.013–0.042) | 1 | 1.140 ± 0.168 | 5.250 | |
| Almora | 24 | 0.390 (0.286–0.501) | 1.969 (1.413–3.266) | 97.50 | 1.820 ± 0.240 | 3.350 |
| 48 | 0.183 (0.128–0.254) | 1.186 (0.717–2.899) | 183.00 | 1.580 ± 0.250 | 1.800 | |
| 72 | 0.077 (0.054–0.105) | 0.463 (0.288–1.060) | 77.00 | 1.640 ± 0.260 | 1.030 | |
| †Fiducial limits are provided in round brackets. | ||||||
| ‡Resistance ratios are calculated relative to the least resistant population. | ||||||
3.1. Comparative susceptibility of different populations of rice and wheat storage pests to phosphine
3.1.1. Sitophilus oryzae
The difference in susceptibility of the adult stage of S. oryzae was evaluated for three locations and three exposure periods (Table 1). It was observed that the Cuttack population was highly susceptible to phosphine with an LC50 value of 0.004 mg/L at an exposure period of 24 hours. The resistance ratios were calculated for the other two populations, i.e., Delhi and Almora by comparing their respective LC50 values with that of Cuttack, and were estimated to be 75.75 and 97.50 respectively at 24-hour exposure period. At an exposure period of 72 h, 0.001–0.077 mg/L was sufficient to achieve 50% kill in all three populations when compared to 0.004–0.390 mg/L at 24 h exposure.
3.1.2. Rhyzopertha dominica
There was considerable variation in the susceptibility levels of the adult stage of R. dominica among the three populations tested (Table 2). The lowest LC50 of 0.006 mg/L was recorded for the Cuttack population at an exposure period of 24 hours. The LC50 values were in the range of 0.006-1.1 mg/L for the three populations tested at 24-hour exposure period, but a trend of decrease in the LC50 values to the range of 0.003–0.032 mg/L was observed as the exposure period was increased to 72 hours. The Almora population was found to be 183.33 times resistant and the Delhi population was 9.17 times resistant than the Cuttack population, when the LC50 values were compared at 24-hour exposure period.
| Location | Exposure period (hrs) | LC50 (95% Fiducial limit†) (mg/L) | LC95 (95% Fiducial limit†) (mg/L) | Resistance Ratio‡ | Slope ± SE | χ2 calc. |
|---|---|---|---|---|---|---|
| Delhi | 24 | 0.055 (0.032–0.085) | 2.686 (1.108–13.267) | 9.17 | 0.974 ± 0.150 | 2.150 |
| 48 | 0.047 (0.028–0.070) | 1.618 (0.755–6.047) | 11.75 | 1.070 ± 0.160 | 2.467 | |
| 72 | 0.032 (0.018–0.049) | 0.899 (0.457–2.878) | 10.67 | 1.138 ± 0.170 | 2.687 | |
| Cuttack | 24 | 0.006 (0.003–0.010) | 0.208 (0.102–0.701) | 1 | 1.088 ± 0.169 | 3.653 |
| 48 | 0.004 (0.002–0.007) | 0.105 (0.057–0.283) | 1 | 1.173 ± 0.174 | 4.491 | |
| 72 | 0.003 (0.001–0.005) | 0.069 (0.039–0.175) | 1 | 1.202 ± 0.179 | 5.155 | |
| Almora | 24 | 1.100 (0.907–1.311) | 3.997 (2.963–6.540) | 183.33 | 2.280 ± 0.320 | 5.760 |
| 48 | 0.733 (0.586–0.884) | 2.349 (1.805–4.825) | 183.25 | 2.490 ± 0.370 | 6.560 | |
| 72 | 0.077 (0.008–0.197) | 0.463 (0.288–1.060) | 25.67 | 1.460 ± 0.210 | 5.670 | |
| †Fiducial limits are provided in round brackets. | ||||||
| ‡Resistance ratios are calculated relative to the least resistant population. | ||||||
3.1.3. Tribolium castaneum
The relative susceptibility levels of three life stages, viz. egg, larva, and adult of T. castaneum to phosphine at 3 exposure periods were assessed (Table 3). The lethal concentration values sufficient to kill 50% of the population were highest in the case of egg stage which ranged from 0.891–1.979 mg/L among the three populations at 24 hours of exposure. The LC50 values for larval and adult stages at an exposure period of 24 hours were in the ranges of 0.046–0.429 mg/L and 0.227–0.478 mg/L respectively, which were lower than that of the egg stage. There was a reduction in the concentration needed to cause 50% kill in all three stages tested at 72 hours of exposure with LC50 values ranging from 0.114–1.113, 0.009–0.327, and 0.062–0.356 for egg, larva, and adult stages respectively. The most susceptible was the Almora population and hence was used as the base population to determine the resistant ratio of the other two populations. When compared to the other populations, the Cuttack population was found to be the resistant one, with resistance ratios of 2.22, 9.33, and 2.10 for egg, larva, and pupa, respectively at an exposure period of 24 hours.
| Location | Insect life stage | Exposure period (hrs) | LC50 (95% Fiducial limit†) (mg/L) | LC95 (95% Fiducial limit†) (mg/L) | Resistance Ratio‡ | Slope ± SE | χ2 calc. |
|---|---|---|---|---|---|---|---|
| Delhi | Egg | 24 | 1.020 (0.474–1.359) | 4.665 (2.734–46.217) | 1.14 | 2.280 ± 0.320 | 5.760 |
| 48 | 1.010 (0.304–1.015) | 3.087 (2.026–15.222) | 3.13 | 2.785 ± 0.471 | 9.651 | ||
| 72 | 0.837 (0.482–1.224) | 3.087 (2.026–15.220) | 7.34 | 3.058 ± 0.483 | 11.888 | ||
| Larva | 24 | 0.121 (0.041–0.360) | 15.760 (5.290–46.900) | 2.63 | 0.836 ± 0.242 | 0.653 | |
| 48 | 0.081 (0.055–0.115) | 1.675 (0.859–4.921) | 6.23 | 1.248 ± 0.164 | 2.574 | ||
| 72 | 0.059 (0.034–0.096) | 1.224 (0.542–5.970) | 6.55 | 1.253 ± 0.170 | 5.162 | ||
| Adult | 24 | 0.238 (0.085–0.669) | 20.114 (5.150–46.540) | 1.05 | 0.882 ± 0.105 | 0.751 | |
| 48 | 0.122 (0.038–0.393) | 22.990 (7.140–74.090) | 1.08 | 0.737 ± 0.259 | 0.859 | ||
| 72 | 0.073 (0.028–0.192) | 4.810 (1.840–12.590) | 1.18 | 0.937 ± 0.213 | 0.582 | ||
| Cuttack | Egg | 24 | 1.979 (1.565–2.462) | 12.361 (8.756–20.168) | 2.22 | 2.067 ± 0.216 | 8.687 |
| 48 | 1.446 (1.100-1.846) | 11.999 (8.120–21.090) | 4.48 | 1.789 ± 0.195 | 8.167 | ||
| 72 | 1.113 (0.829–1.435) | 9.564 (6.486–16.772) | 9.76 | 1.760 ± 0.195 | 7.992 | ||
| Larva | 24 | 0.429 (0.341–0.511) | 1.734 (1.423–2.273) | 9.33 | 2.714 ± 0.290 | 4.979 | |
| 48 | 0.363 (0.284–0.436) | 1.512 (1.242–1.987) | 27.92 | 2.652 ± 0.288 | 6.665 | ||
| 72 | 0.327 (0.251–0.397) | 1.273 (1.047–1.664) | 36.33 | 2.783 ± 0.316 | 7.964 | ||
| Adult | 24 | 0.478 (0.381–0.567) | 1.785 (1.477–2.313) | 2.10 | 2.872 ± 0.309 | 6.456 | |
| 48 | 0.407 (0.316–0.492) | 1.650 (1.350–2.173) | 3.60 | 2.707 ± 0.301 | 8.791 | ||
| 72 | 0.356 (0.271–0.435) | 1.453 (1.187–1.916) | 5.74 | 2.695 ± 0.308 | 6.311 | ||
| Almora | Egg | 24 | 0.891 (0.640–1.181) | 6.570 (3.624–25.580) | 1 | 11.470 ± 0.310 | 4.355 |
| 48 | 0.323 (0.140–0.489) | 4.823 (1.785–12.186) | 1 | 9.270 ± 0.250 | 5.127 | ||
| 72 | 0.114 (0.041–0.333) | 2.017 (0.873–5.082) | 1 | 4.890 ± 0.220 | 4.826 | ||
| Larva | 24 | 0.046 (0.030–0.073) | 0.305 (0.166–0.908) | 1 | 1.560 ± 0.260 | 1.010 | |
| 48 | 0.013 (0.008–0.054) | 0.058 (0.034–0.160) | 1 | 11.930 ± 0.350 | 0.470 | ||
| 72 | 0.009 (0.002–0.020) | 0.028 (0.020–0.059) | 1 | 2.580 ± 0.410 | 1.430 | ||
| Adult | 24 | 0.227 (0.157–0.314) | 1.741 (1.060–3.979) | 1 | 1.440 ± 0.210 | 0.830 | |
| 48 | 0.113 (0.076–0.160) | 1.021 (0.591–2.554) | 1 | 1.340 ± 0.190 | 1.520 | ||
| 72 | 0.062 (0.045–0.083) | 0.271 (0.174–0.647) | 1 | 1.990 ± 0.370 | 0.380 | ||
| †Fiducial limits are provided in round brackets. | |||||||
| ‡Resistance ratios are calculated relative to the least resistant population. | |||||||
3.1.4. Trogoderma granarium
The larval stage of three populations of T. granarium was subjected to phosphine bioassay at 3 exposure periods to determine the relative susceptibility levels (Table 4). Delhi population proved to be the most susceptible population among the three with the lowest LC50 value of 0.242 mg/L at 24-hour exposure period. The most resistant population turned out to be Almora with a resistance ratio of 10.72 when the LC50 values were determined for an exposure period of 24 hours. The LC50 values which were in the range of 0.242–2.594 mg/L at 24 hours exposure period were found to have reduced to the range of 0.050–0.682 mg/L when the exposure period was increased to 72 hours.
| Location | Exposure period (hrs) | LC50 (95% Fiducial limit†) (mg/L) | LC95 (95% Fiducial limit†) (mg/L) | Resistance Ratio‡ | Slope ± SE | χ2 calc. |
|---|---|---|---|---|---|---|
| Delhi | 24 | 0.242 (0.099–0.590) | 11.350 (4.650–27.730) | 1 | 0.995 ± 0.198 | 0.910 |
| 48 | 0.106 (0.036–0.313) | 11.990 (4.060–35.440) | 1 | 0.812 ± 0.240 | 0.975 | |
| 72 | 0.050 (0.014–0.181) | 6.660 (1.830–24.220) | 1 | 0.788 ± 0.286 | 0.898 | |
| Cuttack | 24 | 0.641 (0.315–0.931) | 3.180 (1.860–16.220) | 2.650 | 2.365 ± 0.511 | 11.607 |
| 48 | 0.493 (0.359–0.614) | 2.558 (1.892–4.192) | 4.650 | 2.299 ± 0.332 | 9.570 | |
| 72 | 0.402 (0.288–0.506) | 1.843 (1.415–2.779) | 8.040 | 2.489 ± 0.351 | 7.146 | |
| Almora | 24 | 2.594 (2.225–3.037) | 5.536 (4.350–8.897) | 10.720 | 3.890 ± 0.710 | 0.850 |
| 48 | 0.938 (0.730–1.145) | 2.141 (1.617–4.155) | 8.850 | 3.580 ± 0.830 | 2.343 | |
| 72 | 0.682 (0.532–0.839) | 1.732 1.318–2.827) | 13.640 | 3.160 ± 0.560 | 5.619 | |
| †Fiducial limits are provided in round brackets. | ||||||
| ‡Resistance ratios are calculated relative to the least resistant population. | ||||||
| Hypothenemus hampei | Larva | 24 | 0.015 (0.009–0.022) | 0.095 (0.049–0.553) | 2.045 ± 0.226 | 18.946 |
| 48 | 0.0049 (0.0023–0.0083) | 0.0313 (0.0149–0.4792) | 2.053 ± 0.232 | 22.260 | ||
| 72 | 0.0033 (0.0018–0.0048) | 0.0213 (0.0123–0.082) | 2.032 ± 0.167 | 24.371 | ||
| Pupa | 24 | 0.024 (0.019–0.033) | 0.169 (0.090–0.604) | 1.932 ± 0.249 | 7.5078 | |
| 48 | 0.0071 (0.0046–0.0115) | 0.0588 (0.0262–0.6646) | 1.795 ± 0.236 | 11.801 | ||
| 72 | 0.0048 (0.0029–0.0068) | 0.0234 (0.0139–0.0806) | 2.387 ± 0.179 | 28.585 | ||
| Adult | 24 | 0.021 (0.018–0.025) | 0.058 (0.043–0.103) | 3.805 ± 0.305 | 16.245 | |
| 48 | 0.0066 (0.0043–0.0101) | 0.0457 (0.0222–0.3522) | 1.955 ± 0.170 | 25.665 | ||
| 72 | 0.0038 (0.0021–0.0055) | 0.0188 (0.0113–0.0644) | 2.348 ± 0.171 | 31.718 | ||
| Callosobruchus maculatus | Egg | 24 | 1.915 (1.563–2.773) | 12.050 (5.980–80.890) | 2.060 ± 0.493 | 1.465 |
| 48 | 1.620 (1.360–2.050) | 8.370 (4.870–29.860) | 2.309 ± 0.486 | 2.493 | ||
| 72 | 1.550 (1.310–1.890) | 7.180 (4.450–20.720) | 2.469 ± 0.490 | 3.170 | ||
| †Fiducial limits are provided in round brackets | ||||||
3.2 Comparative toxicity of phosphine against Hypothenemus hampei and Callosobruchus maculatus
Three life stages of H. hampei, viz. larva, pupa, and adult, and one life stage of C. maculatus, i.e. egg were subjected to phosphine bioassay at 24, 48 and 72 hours of exposure periods to determine the efficacy of phosphine against these storage pests (Table 5). Phosphine at 0.2 mg/L could give more than 90% mortality of H. hampei under laboratory bio-assay studies. LC50 values in the range of 0.015–0.024 mg/L at 24-hour exposure period against larval, pupal, and adult stages of the insect proved their high susceptibility to the fumigant. The highest LC50 of 0.024 mg/L was observed in the case of the pupal stage of H. hampei and the lowest of 0.015 mg/L was recorded in the larval stage. It was also noticed that as in the case of other storage insects tested, there was a downward trend of LC50 values in all three stages tested when the exposure period was increased to 48 hours and then to 72 hours. The laboratory bioassays with the egg stage of C. maculatus revealed the LC50 values to be in the range of 1.550–1.915 mg/L for the different exposure periods of 24, 48, and 72 hours. The decreasing trend of LC50 values with the increase in exposure period was invariably noticed in the case of C. maculatus.
As with other control agents, long-term heavy reliance on phosphine under circumstances where treatments are inadequate, leads to selection of resistance in pest populations (Benhalima, Chaudhry, Mills, & Price, 2004). Repeated under-dosing, inadequate exposure periods, and less airtight conditions had been shown as causes for a gradual increase in the populations showing resistance to phosphine (Champ & Dyte, 1976). Work characterizing weak and strong phosphine resistance phenotypes finds RR50 values for weakly resistant populations to be less than 50, while strongly resistant populations may have RR50 values from 50 to over 1,000 depending on the type of dose-mortality trial conducted (Nayak, Kaur, Jagadeesan, Pavic, Phillips & Daglish, 2019). Studies show that increasing either concentration or time will increase efficacy against resistant insects, but the effects are unequal, with time usually being more important than the concentration (Collins, Daglish, Pavic & Kopittke, 2005; Daglish, Collins, Pavic & Kopittke, 2002; Kaur & Nayak, 2015). An important biological factor affecting phosphine efficacy is the insect developmental stage. Eggs and pupae of resistant insects tend to be more tolerant to phosphine than larvae or adults (Hole, Bell, Mills & Goodship, 1976; Manivannan, 2015; Venkidusamy et al. 2018).
Previous reports by Rajendran (1999), Ling (1999), and Nguyen, Collins & Ebert (2015) indicated the development of phosphine resistance in populations of S. oryzae from India, China, and Vietnam respectively. In the present study, we could observe that among the three populations of S. oryzae tested, Almora was found to be more resistant which was 97.50 times more resistant than the susceptible population from Cuttack at 24 hours exposure period. Since the resistance factor is more than 50, the population from Almora was designated as a strongly resistant population in comparison to the Cuttack population. Similarly, Nguyen et al. (2015) also designated one of the strains in their study as a strongly resistant strain which had an LC50 52 times greater than a susceptible reference strain. The susceptible population of S. oryzae from Cuttack recorded one of the lowest LC50 values (0.004 mg/L) at 24-hour exposure period among all the test insects subjected to bioassay in this study. The only stage/insect with a lower LC50 value than the Cuttack population of S. oryzae was the larval stage of H. hampei. As the exposure period increased, there was a reduction in the LC50 values of populations of S. oryzae. But an exception was noticed in the Cuttack population which was comparatively more susceptible. The LC50 value was reduced when the exposure period was increased from 24 hours to 48 hours. But, when the exposure period was increased to 72 hours, the LC50 value remained the same. This can be attributed to the fact that the LC50 value for the Cuttack population at 48-hour exposure period was too low and the difference in the mortality data in the 72-hour exposure period was not enough to make a change in the LC50 value. However, there was a slight change in the fiducial limits when the exposure period increased from 48 hours to 72 hours. The trend of LC95 values reducing with the increased exposure period further added weightage to the inference that the increase in exposure periods results in a decrease in the lethal concentration values.
The adult stage of the three populations of R. dominica were tested for susceptibility to phosphine and it was found that the Almora population was the most resistant one with a resistance ratio as high as 183.33 at an exposure period of 24 hours when compared to the susceptible population at Cuttack. So, the Almora population was characterized as a strongly resistant one, while the population from Delhi with a resistance ratio of 9.17 at 24 hours exposure period was designated as weakly resistant. We could also observe that all the lethal concentration values, viz. LC50, LC90 and LC95 tend to decrease as the exposure periods are increased. Rhyzopertha dominica was the first pest in which strong resistance to phosphine was recorded in Australia (Collins, 1998). Opit et al. (2012) studied phosphine resistance in T. castaneum and R. dominica in stored wheat in Oklahoma and found that the most resistant T. castaneum population was 119-fold more resistant than the susceptible strain and the most resistant R. dominica population was over 1,500-fold more resistant. We could also observe similar results with the most resistant R. dominica population with a higher resistance ratio of 183.33 when compared to that of T. castaneum (2.10), and the resistance ratio of 183.33 at 24-hour exposure period was the highest among all the storage pests tested in this study. We presume that the resistance ratios might have been much higher if these field populations could have been compared with a laboratory-susceptible population that had not been exposed to the fumigant for several years.
Significant variations in the susceptibility levels to phosphine and the development of resistance in different populations of T. castaneum were reported from various parts of the world (Acda, 2000; Cato et al. 2017; Gautam et al. 2016; Kaur et al. 2015; Mills, 1983; Rajendran, 1999; Ramya et al. 2018; Ren et al. 1994). Phosphine resistance in immature stages in laboratory-selected strains of T. castaneum has been reported by Nakakita & Winks (1981). A few reports also mentioned about exposure to phosphine delaying the development of immature stages of T. castaneum (Rajendran, 2000; Rajendran, Nayak & Anjum SS, 2001). In our study, we have tested three life stages, egg, larva, and adult of three populations to assess whether the susceptibility to phosphine varies among the developmental stages and also among the populations. Among the three stages tested, it was the egg stage which was found to be the least susceptible in all the three populations, followed by adult and larva. Also, among the three populations tested, the Cuttack population which had the highest LC50 values and resistance ratios of 2.22, 9.33, and 2.10 for egg, larva, and adult respectively when compared to the relatively susceptible Almora population at 24-hour exposure period was designated as a weakly resistant strain. Similar to our results, Gautam et al. (2016) who studied phosphine resistance in adult and immature life stages of T. castaneum could infer that the LC50 value for the egg stage was comparatively higher than that of the adults. However, Manivannan (2015) who studied the toxicity of phosphine on the developmental stages of T. castaneum over a range of concentrations and exposures inferred that among the life stages tested, pupae were more tolerant to phosphine followed by the egg and the larval instars.
The experiments to assess the variability in susceptibility of larvae of T. granarium among three populations could reveal that comparatively higher concentrations were required to attain the 50% kill in T. granarium when compared to that of the other storage pests evaluated. The LC50 value of the most resistant population at 24 hours exposure period was 2.594 mg/L which was the highest among all the storage insect pests tested in this study. When Yadav et al. (2020) assessed phosphine resistance in populations of T. granarium collected from North India, the highest LC50 value for the most resistant population was 1.012 mg/L and the resistance ratio was 31.62. Since we couldn’t compare our most resistant population with a laboratory-susceptible population, the resistance ratio (10.272) was not as high as that of Yadav et al. (2020). However, the LC50 value of the most resistant population in our study (2.594 mg/L) being 2.5 times higher than that of Yadav et al. (2020) reiterates the urgent need to look into corrective measures as T. granarium is classified among the 100 worst invasive species worldwide (Harris, 2006). We could also observe the general trend of a decrease in LC50 values with an increase in exposure periods in the case of T. granarium too.
There are very few studies done to determine the efficacy of phosphine against C. maculatus. In one of our previous studies, phosphine efficacy was investigated against laboratory cultured and field populations of C. maculatus by organizing field fumigation trials and could observe no emergence of adults until 60 days after treatments (2 tab/MT, 3 tab/MT, 1.0 g and 1.5 g phosphine/m3) indicating 100% insect mortality (Arora & Srivastava, 2021). Similarly, Aidbhavi et al. (2022) attempted to the comparative assessment of phosphine toxicity to the developmental stages of bruchid species in India and inferred that the egg stage of C. maculatus was comparatively less sensitive to phosphine. Our results were concordant with the previous studies as the LC50 value for the egg stage at 24 hours exposure period was 1.915 mg/L which was higher in comparison to that of the other storage insects tested in this study. The only other storage insect with a higher LC50 than that of the eggs of C. maculatus is the larval stage of the most resistant population of T. granarium (2.594 mg/L) and the egg stage of the most resistant population of T. castaneum (1.979 mg/L).
There is no previous information available on the efficacy of phosphine against the coffee berry borer, H. hampei. Angelica et al. (2019) reported the efficacy of cyantraniliprole against adults of H. hampei with a lethal concentration of 0.67 mg/ml. However, under present studies, phosphine is observed to be effective against larval, pupal, and adult stages of H. hampei with lethal concentration in the range 0.015–0.024 mg/L at 24 hours of exposure period. It was also observed that coffee berry borer is comparatively more susceptible to phosphine when compared to other stored grain pests. This can be attributed to the fact that these insects are comparatively less exposed to phosphine in comparison to storage pests of cereals and pulses. It was also learned that the LC50 and LC90 values tend to decrease with increased exposure periods, which underscores the importance of extended exposure periods rather than increased doses for better management of stored grain pests including CBB using phosphine. We could also understand the order of susceptibility of life stages to phosphine, which was larvae > adult > pupae. This can be attributed to the reduced respiratory rates in pupae which led to reduced intake of phosphine, which is a fumigant (Chaudhry, 1997).
Most of the wheat storage facilities in India are not gas-tight, and such leakiness is probably the main cause of under-dosing (low gas concentration and short exposure times) and consequent survival of insect pests. The practice of over-dosing structures to compensate for the inability to adequately seal these structures before fumigation is common in India and could also have contributed to an increase in resistance in the storage pests tested in this study. The insecticidal action of phosphine is different from that of other fumigants as the relationship of its dosage with concentration and the time of exposure is not linear. Longer exposure time has often proved to be more effective than higher concentrations alone. Higher concentrations can induce a state of ‘narcosis’ in most species of stored product insects, which may protect them from toxic effects. So, increased exposure periods might be the better option for the efficient management of storage pests using phosphine.
Considerable variation in susceptibility to phosphine was observed among the various populations of the six storage insect pests tested. Our study also reveals that among the six storage pests studied, the LC50 values decreased for all of them as the exposure periods increased. Since resistant insects can survive higher concentrations over a limited period, but not relatively low concentrations over long exposure periods, the main strategy to achieve maximum effectiveness of phosphine against resistant insect pests must, therefore, be to aim for extended exposure periods. Despite the high levels of resistance, a combination of optimal doses of phosphine and extended exposures can still provide effective control of these pests. The evolution of resistance to phosphine as reported by this study in T. granarium, which is a notorious quarantine pest warrants further evaluation to assess the level of resistance in wider geographical areas and also calls for deliberations to design appropriate fumigation strategies to overcome the concerns caused by the development of phosphine resistance. This study also could bring to light that phosphine can be an efficient fumigant against the various developmental stages of H. hampei. Further, surveying the history of pest management practices in facilities where insects tested in this study originated will elucidate factors contributing to the development of PH3 resistance in insects and mitigating resistance development.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study was supported and funded by the Department of Agriculture and Cooperation (DAC), Ministry of Agriculture and Farmers Welfare, Govt. of India (Grant No. 8–35/2017-PP-II). The authors are thankful to DAC, Ministry of Agriculture and Farmers Welfare, Govt of India for funding this project; and Dr PK Chakrabarty, ASRB, Member and Ex-ADG (PP and BS), ICAR for facilitating the research activities under this project. We are also thankful to Mr. Ujjwal Kumar, Business Head, and his team, UPL India Pvt Ltd for helping in arranging the infrastructure, and Dr S. Rajendran, Ex-Scientist, CSIR-CFTRI and Directors of all ICAR Institutes at different locations for extending their support in successful completion of the research activities.
Authors’ contributions
SA conceptualized the project. SA designed the research with inputs from CS and RSR. RSR, ON, JS, NP, TA, MJ, JPG, and DP conducted and performed all experiments. SA compiled and analyzed the data. RSR wrote the manuscript with contributions from SA and JS. All authors read and approved the manuscript.
- Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265-267.
- Acda MA (2000) Phosphine resistance in Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) from the Philippines. MS Thesis, University of Queensland, Brisbane, Australia
- Adriano EP, Gontijo PC, Fantine AK, Tinoco RS, Ellersieck MR, Carvalho GA, Zanuncio JC, Vilela EF (2021) Emergence and infestation level of Hypothenemus hampei (Coleoptera: Curculionidae) on coffee berries on the plant or on the ground during the post-harvest period in Brazil. J Insect Sci 21(2):10.
- Afful E, Brent E, Nayak MK, Thomas PW (2018) Phosphine resistance in North American field populations of the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae). J Econ Entomol 111(1):463-469.
- Aidbhavi R, Subramanian S, Ranjitha MR, Chandel RK, Srivastava C, Bandi SM, Singh B (2022) Comparative toxicity of phosphine to developmental stages of three Callosobruchus species infesting stored pulses. J Asia Pac Entomol 25:101871.
- Angelica PR, Martínez LC, Da Silva BKR, Zanuncio JC, Fernandes MES, Guedes RNC, Fernandes FL (2019) Exposure to cyantraniliprole causes mortality and disturbs behavioral and respiratory responses in the coffee berry borer (Hypothenemus hampei). Pest Manag Sci 75(8):2236-2241.
- Arora S, Srivastava C (2021) Locational dynamics of concentration and efficacy of phosphine against pulse beetle, Callosobruchus maculatus (Fab). Crop Prot 143:105475.
- Arora S, Stanley J, Srivastava C (2021) Temporal dynamics of phosphine fumigation against insect pests in wheat storage. Crop Prot 144:105602.
- Athanassiou CG, Phillips TW, Wakil W (2019) Biology and control of the khapra beetle, Trogoderma granarium, a major quarantine threat to global food security. Annu Rev Entomol 64:131-148.
- Benhalima H, Chaudhry MQ, Mills KA, Price NR (2004) Phosphine resistance in stored-product insects collected from various grain storage facilities in Morocco. J Stored Prod Res 40:241-249.
- Bhatia SK, Pradhan S (1968) Studies on resistance to insecticides in Tribolium castaneum (Herbst). III. Selection of a strain resistant to p,p’ DDT and its biological characteristics. Indian J Entomol 30:13-32.
- Brun LO, Gaudichon V, Wigley PJ (1993) An artificial diet for continuous rearing of the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae). Insect Sci Appl 14(5/6):585-587.
- CABI (2019) Invasive Species Compendium. Available via DIALOG. https://www.cabi.org/ISC. Accessed 07 Mar 2022.
- Cao Y, Son Y, Sun GY (2003) A survey of psocid species infesting stored grain in China and resistance to phosphine in field populations of Liposcelis entomophila (Enderlein) Psocoptera: Liposcelididae). In Proceedings of the 8th International Working Conference on Stored Product Protection (pp. 662-667). Wallingford, United Kingdom.
- Cato AJ, Elliott B, Nayak MK, Phillips TW (2017) Geographic variation in phosphine resistance among North American populations of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). J Econ Entomol 110:1359-1365.
- Champ BR, Dyte CE (1976) Report of the FAO global survey of pesticide susceptibility of stored grain pests. FAO Plant Production and Protection Series 5:260-297.
- Chaudhry MQ (1997) A review of the mechanisms involved in the action of phosphine as an insecticide and phosphine resistance in stored-product insects. Pestic Sci 49(3):213-228.
- Chen Z, Schlipalius D, Opit G, Subramanyam B, Phillips TW (2015) Diagnostic molecular markers for phosphine resistance in U.S. populations of Tribolium castaneum and Rhyzopertha dominica. PLoS One 10(3):e0121343.
- Collins PJ (1998) Resistance to grain protectants and fumigants in insect pests of stored products in Australia. In: Proceedings of the Australian Post-harvest Technical Conference (pp. 55-57). CSIRO, Canberra, Australia.
- Collins PJ, Daglish GJ, Bengston M, Lambkin TM, Pavic H (2002) Genetics of resistance to phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae). J Econ Entomol 95:862-869.
- Collins PJ, Daglish GJ, Pavic H, Kopittke RA (2005) Response of mixed age cultures of phosphine-resistant and susceptible strains of lesser grain borer, Rhyzopertha dominica to phosphine at a range of concentrations and exposure periods. J Stored Prod Res 41:373-385.
- Daglish GJ, Collins PJ, Pavic H, Kopittke KA (2002) Effects of time and concentration on mortality of phosphine-resistant Sitophilus oryzae (L.) fumigated with phosphine. Pest Manag Sci 58:1015-1021.
- EPPO (European and Mediterranean Plant Protection Organization) (2022) EPPO Global Data Base. Trogoderma granarium. Available via DIALOG. https://gd.eppo.int/taxon/TROGGA. Accessed 18 Feb 2022.
- FAO (1975) Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides: tentative method for adults of some stored cereals, with methyl bromide and phosphine: FAO Method No. 16. FAO Plant Prot Bull 23:12-25.
- FAOSTAT (2019) Food and Agriculture Organization of the United Nations, Statistics Division. Available via DIALOG. http://faostat.fao.org/. Accessed 18 Feb 2022.
- Gautam SG, Opit GP, Hosoda E (2016) Phosphine resistance in adult and immature life stages of Tribolium castaneum (Coleoptera: Tenebrionidae) and Plodia interpunctella (Lepidoptera: Pyralidae) populations in California. J. Econ Entomol 109: 2525-2533.
- Gautam SG, Opit GP, Konemann C, Shakya K, Hosoda E (2020) Phosphine resistance in saw-toothed grain beetle, Oryzaephilus surinamensis in the United States. J Stored Prod Res 89:101690.
- Gustavsson J, Cederberg C, Sonesson U, van Otterdijk R, Meybeck A (2011) Global food losses and food waste- extent, causes and prevention. FAO, Rome.
- Hagstrum DW, Phillips TW, Cuperus GW (2012) Stored Product Protection. Kansas State University, Manhattan, USA.
- Harris DL (2006) Khapra Beetle, Trogoderma granarium Evert (Insecta: Coleoptera: Dermestidae). University of Florida, IFAS Extension (EENY-372 (IN667).8.
- Hole BD, Bell CH, Mills KA, Goodship G (1976) The toxicity of phosphine to all developmental stages of thirteen species of stored product beetles. J Stored Prod Res 12:235-244.
- Kaur R, Nayak MK (2015) Developing effective fumigation protocols to manage strongly phosphine resistant Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae). Pest Manag Sci 71:1297-1302.
- Kaur R, Subbarayalu M, Jagadeesan R, Daglish GJ, Nayak MK, Naik HR, Ramasamy S, Subramanian C, Ebert PR, Schlipalius DI (2015) Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes. Heredity 115(3):1-7.
- Kavallieratos NG, Athanassiou CG, Boukouvala MC, Tsekos GT (2019) Influence of different non-grain commodities on the population growth of Trogoderma granarium Everts (Coleoptera: Dermestidae). J Stored Prod Res 81:31-39.
- Kumar U (2018) Sustained effective use of phosphine in stored product protection in India: Role of UPL Limited. In Proceedings of 12th International Working Conference on Stored Product Protection (pp. 952-959). Berlin, Germany.
- Ling Z (1999) Development and countermeasures of resistance in stored grain insects in Guangdong of China. In Proceedings of the 7th International Working Conference on Stored Product Protection (pp. 642-647). Chengdu, China.
- Lorini I, Collins PJ, Daglish GJ, Nayak MK, Pavic H (2007) Detection and characterization of strong resistance to phosphine in Brazilian Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). Pest Manag Sci 63:358-364.
- Manivannan S (2015) Toxicity of phosphine on the developmental stages of rust-red flour beetle, Tribolium castaneum Herbst over a range of concentrations and exposures. J Food Sci Technol 52(10):6810-6815.
- Miller A, Phillips R, Cline LD (1969) Rearing manual for stored-product insects used by USDA Stored-Product Insects Research and Development Laboratory, Savannah, GA.
- Mills KA (1983) Resistance to the fumigant hydrogen phosphide in some stored product insect species associated with repeated inadequate treatments. Mitteilungen der Deutschen Gesellschaft fur Allgemeine und Angewandte Entomologie 4:98-101.
- Monro HAU, Musgrave AJ, Upitis E (1961) Induced tolerance of stored product beetles to methyl bromide. Ann Appl Biol 49:373-377.
- Myers SW, Hagstrum DW (2012) Quarantine. In: DW Hagstrum, TW Phillips & G Cuperus (Eds.), Stored Product Protection (pp. 297-304). Kansas State University, Manhattan, USA,
- Nakakita H, Winks RG (1981) Phosphine resistance in immature stages of a laboratory selected strain of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). J Stored Prod Res 17:43-52.
- Nakakita H (1998) Stored rice and stored product insects. In: H. Nakakita (Ed.), Rice Inspection Technology (pp. 49-65). ACE Corporation, Tokyo.
- Nayak MK, Collins PJ, Holloway JC, Emery RN, Pavic H, Bartlet J (2013) Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae): Its characterization, a rapid assay for diagnosis and its distribution in Australia. Pest Manag Sci 69:48-53.
- Nayak MK, Daglish GJ, Phillips TW, Ebert PR (2020) Resistance to the fumigant phosphine and its management in insect pests of stored products: a global perspective. Annu Rev Entomol 65:333-350.
- Nayak MK, Kaur R, Jagadeesan R, Pavic H, Phillips TW, Daglish GJ (2019) Development of a quick knock down test for diagnosing resistance to phosphine in rice weevil, Sitophilus oryzae, a major pest of stored products. J Econ Entomol 112(4):1975-1982.
- Nguyen TT, Collins PJ, Ebert PR (2015) Inheritance and characterization of strong resistance to phosphine in Sitophilus oryzae (L.). Plos One 10(4):e0124335.
- Nguyen TT, Collins PJ, Duong TM, Schlipalius DI, Ebert PR (2016) Genetic conservation of phosphine resistance in the rice weevil Sitophilus oryzae (L.). J Hered 107:228-237.
- Opit G, Phillips T, Aikins M, Hasan M (2012) Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma. J Econ Entomol 105:1107-1114.
- Perez‐Mendoza J, Dover BA, Hagstrum DW, Hopkins TL (1999) Effect of crowding, food deprivation, and diet on flight initiation and lipid reserves of the lesser grain borer, Rhyzopertha dominica. Entomol Exp Appl 91(2):317-326.
- Peter AF (2018) Irradiation for quarantine control of coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) in coffee and a proposed generic dose for snout beetles (Coleoptera: Curculionoidea). J Econ Entomol 111(4):1633-1637.
- Pike V (1994) Laboratory assessment of the efficacy of phosphine and methyl bromide fumigation against all life stages of Liposcelis entomophilus (Enderlein). Crop Prot 13:141-145.
- Rajashekar Y, Gunasekaran N, Shivanandappa T (2010) Insecticidal activity of the root extract of Decalepis hamiltonii against stored-product insect pests and its application in grain protection. J Food Sci Technol 47(3):310-314.
- Rajendran S (1994) Psocids in food commodities and their control. Pestology 28:14-19.
- Rajendran S (1999) Phosphine resistance in stored grain insect pests in India. In Proceedings of Seventh International Working Conference on Stored Product Protection (pp. 635-641). Chengdu, China.
- Rajendran S (2000) Inhibition of hatching of Tribolium castaneum by phosphine. J Stored Prod Res 36:101-106.
- Rajendran S, Nayak KR, Anjum SS (2001) The action of phosphine against the eggs of phosphine-resistant and -susceptible strains of Rhyzopertha dominica F. Pest Manag Sci 57:422-426.
- Ramya RS, Srivastava C, Subramanian S (2018) Monitoring of phosphine resistance in Indian populations of Tribolium castaneum (Herbst) from stored wheat. Indian J Entomol 80(1):19-23.
- Ramya RS, Srivastava C, Subramanian S, Ranjith M (2023) Inheritance pattern and expression of resistance to phosphine in larval stage of Tribolium castaneum (Coleoptera: Tenebrionidae). J Asia Pac Entomol 26(1):102040.
- Ren YL, O’Brien IG, Whittle GP (1994) Studies on the effect of carbon dioxide in insect treatment with phosphine. In Proceedings of the 6th International Working Conference on Stored Product Protection (pp. 173-177). Wallingford, United Kingdom.
- Venkidusamy M, Jagadeesan R, Nayak MK, Subbarayalu M, Subramanian C, Collins PJ (2018) Relative tolerance and expression of resistance to phosphine in life stages of the rusty grain beetle, Cryptolestes ferrugineus. J Pest Sci 91:277-286.
- Winks RG (1986) The effect of phosphine on resistant insects. In Proceedings of the GASCA Seminar on Fumigation Technology in Developing Countries (pp. 105-118). London.
- Yadav SK, Srivastava C, Subramanian S (2020) Phosphine resistance and antioxidant enzyme activity in Trogoderma granarium Everts. J Stored Prod Res 87:101636.