There is an urgent need for the development of a standardized protocol for resistance to phosphine, that can be widely adopted by the scientific community. This protocol should be reliable, but at the same time easy to use and perhaps less laborious, as the industry usually does not have access to specialized laboratories, or when there is an access, resistance has been already well developed. 

Christos G. ATHANASSIOU

Laboratory of Entomology and Agricultural Zoology Department of Agriculture, Crop Production and Rural Environment University of Thessaly, Greece

Current status The phase-out of methyl bromide (UNEP, 1995) has increased significantly the reliance on phosphine, as a fumigant for stored-product protection (Bell, 2000; Chaudhry, 2000; Nayak, 2012). Phosphine (PH3) is the most commonly used aerial insecticide for the control of stored product insects globally. It is applicable in a wide range of products and foods such as cereals grains, tobacco, dried fruits and other raw and processed foods (Chaundhry, 2000; Nayak and Collins, 2008). Phosphine has been proven to be effective against most major stored product insects and mites, but its extensive and not proper use meets several drawbacks. Repeated applications, poor sealing, high leakages and low concentrations have been reported as responsible for the development of resistance to phosphine in the most major stored product insects and mites (Opit et al., 2012; Nayak et al., 2013). Currently, in contract with some decades ago, where much research was focused on the resistance of stored product insects to organophosphates and pyrethroids, the majority of the published studies on resistance of arthropods at the post-harvest stages of agricultural commodities are almost exclusively on phosphine. Most of the studies available are about the lesser grain borer, Rhyzopertha dominica, the red flour beetle, Tribolium castaneum and more recently, the rusty grain beetle, Cryptolestes ferrugineus, the rice weevil, Sitophilus oryzae and the cigarette beetle, Lasioderma serricorne (Kaur, 2012; Nayak et al., 2013; Saglam et al., 2015; Venkidusamy et al., 2017).

Geographical distribution of resistance to phosphine Based on the published reports so far, resistance to phosphine has been reported in many parts of the world. For the majority of the species tested eggs and pupae are considered as the most resistant life stages. There are numerous reports for resistant insect populations from Brazil (Pimentel et al., 2009; 2010; Lorini et al., 2007), Bangladesh (Tyler et al., 1983), China (Song et al., 2011), Morocco (Benhalima et. al., 2004), India (Rajendran and Narasimhan, 1994; Kaur et al., 2015), Pakistan (Ahmad et al., 2013; Alam et al., 1999), USA (Opit et al., 2012; Chen et al., 2012; Saglam et al., 2015; Konemann et al. 2017; Afful et al., 2017) and Australia (Collins et al. 2005, Nayak and Collins 2008, Emery et al. 2003, 2011, Holloway et al. 2016). In general, much of the work done has been carried out in Australia, where the problem of “strong resistance” is often reported. However, paradoxically, there are not that many published reports from Europe, although phosphine is commonly used in Europe. Hence, the “map of resistance” has wide gaps in several European parts, despite the fact that there are some initial reports of stored product insect populations that are not susceptible to phosphine. The question is: is resistance in Europe that common as it is reported in other countries, such as Australia and Asia? During the last years, the University of Thessaly (Laboratory of Entomology and Agricultural Zoology) has started a thorough surveillance for the evaluation of resistance to phosphine in insect populations from Europe.

Evaluation of resistance One additional variable that constitutes the evaluation of resistance to phosphine a very complicated procedure is the fact that there are several, and often contradictory, methods towards this direction. The question that we often get from several professionals is simple: how can we evaluate resistance to phosphine?

To identify and characterize the resistance of phosphine, various protocols have been used, which may provide different results. There are protocols that are proposed as quick diagnostic tools (less laborious protocols) which can be easily used by the industry, realistic and reliable, and adapted to the industrial scale in “real world” fumigations. One protocol that is used in several parts of the world is the Detia Degesch Phosphine Tolerance Test Kit (DDPTTK) by Steuerwald et al. (2005) (developed by Detia Degesch GmbH in Germany), which is based on a rapid immobilization/narcosis bioassay that can diagnose resistance in a pest population in less than 30 minutes (Fig. 1). DDPTTK is based on the number of insects that are immobilized after certain minutes of exposure to very high concentrations of phosphine (usually at 3000 ppm), and uses immobilization/narcosis as a tolerance indicator. More recently, Nayak et al. (2013) presented a rapid bioassay test, on which insects are exposed to a maximum interval of 5 hours and 15 min. at 1430 ppm or 2 mg/l., which examines immobilization/narcosis of the exposed insects, and based on these characteristics, can differentiate weak from strong resistance.

On the other hand, many research groups throughout the world are based on protocols that are more laborious. Specifically, the Food and Agriculture Organization (FAO) is the most commonly used protocol in the research community. This protocol is usually based on the exposure of insects at low concentrations, such as 30 ppm, for relatively long intervals, such as 20 hours, through the procedure that is described in the FAO Plant Protection Bulletin (FAO, 1975). This protocol has been modified by several scientific groups throughout the globe. The so called Dose Response (or Dose Bioassay) protocol, is practically an advanced FAO procedure: here, the tested insects are placed in jars and exposed to phosphine at different concentrations for a specific exposure time (e.g. 50, 100, 200, 500, 800 and 1000 ppm for 3 or more days). Moreover, the CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) sub-group on pest and Sanitation Management in Stored Tobacco has developed an additional guideline to classify potential resistant strains for L. serricorne and the tobacco moth, Ephestia elutella (CORESTA, 2013). In this regard, the assessment of L. serricorne and E. elutella resistance is operated by using a protocol that lasts between 4 and 12 days, at phosphine concentrations that are between 200 and 700 ppm, depending on the conditions, the target species and strain. Based on the above, it becomes evident that it is quite difficult to compare these protocols. The CORESTA protocol is the strictest among all protocols because has the longest duration of all protocols, which underlines the importance of duration in the commercial facilities (e.g. 6 days exposure). Conversely, DDPTTK is much shorter than the other ones and can provide reliable conclusions in a very short interval, while at the same time can be used on site before the initiation of the fumigation.

Future needs There is an urgent need for the development of a standardized protocol for resistance to phosphine, that can be widely adopted by the scientific community. This protocol should be reliable, but at the same time easy to use and perhaps less laborious, as the industry usually does not have access to specialized laboratories, or when there is an access, resistance has been already well developed. At the same time, expansion of surveillance is a critical parameter that should be taken into account, since there are practically no data for wide areas (incl. Europe) with high levels of production and important export/import activities of durable commodities. In this effort, however, we should bear in mind that not all structures are made for phosphine fumigations, and it is not uncommon to mistakenly consider “failures in fumigation” as “presence of resistance”. Nevertheless, there is an important correlation between those two: failures in fumigations can definitely lead to resistance development.