Detecting insect infestation in soybeans as early as possible is of crucial importance in preventing damage to stored seeds. Various techniques and procedures that can be used to detect the presence of insects in seeds during storage. The following paragraphs describe these different methods, which can be adopted to detect insect infestation in soybean storage. Interested readers are advised to consult Neethirajan et al. (2007) to obtain more details about the detection techniques for stored-product insects in grain.
Visual examination of grain and grain products in bulk or warehouses is a very simple technique. Visual inspection can identify early stages of moth infestations because they make small clusters of seeds or webbing over the surface of seeds or of the storage structure. Such moth larvae are usually restricted to the top portion of the bulk seeds; therefore, they cannot readily be removed by probe traps. Rice weevil larvae feed on the aleurone layer just under the seed coat, which results in a visible pale scar on the outside of the seed. Seeds infested with mature internal-feeding insects become slightly darkened and the kernel is appreciably softened.
Grain probes are generally used to estimate the insect population in grain bins. A grain probe is a hollow metal tube that is inserted into the grain to the desired depth, opened and closed to collect the sample and then removed from the grain and emptied. The sample is then sieved to separate the insects from the seeds. This is a rather laborious method, especially if several locations are sampled to obtain a reliable measure of the numbers and species of insects present.
Probe traps are mainly used for the detection of insects in grains. Loschiavo and Atkinson (1967, 1973) developed a trap that is inserted into seed bulks to collect moving insects. The trap consists of a pointed probe (about 25 mm diameter x 220 mm long) containing glass vials (19 x 65 mm). Above the solid-walled tube is a hollow cylindrical section that is 100 mm long, perforated with 2.2 mm diameter holes. The probe is inserted from the top surface of the seeds with a metal rod. The rod is removed to be used to insert other traps. After 4-7 days (Loschiavo, 1985), the trap is pulled out by a rope tied to the trap. The contents of the trap can be spread onto a flat surface to identify and count any insects and mites that have fallen into the trap.
The number of insects collected in a trap depends on insect mobility, temperature, dockage, the size of the trap perforations, insect species and the length of time the trap is in the seeds. Traps frequently identify infestations that are undetectable with normal seed sampling, because the trap may collect insects from a volume of seeds much larger than the probe. For these reasons, trap counts cannot be used to define the size of an infestation (Muir, 1997).
Detection, and to some degree quantification, of insect infestation in an agricultural commodity is provided by a pitfall probe trap. Loschiavo (1974) designed a modified pitfall trap for use in grain-carrying boxcars. A prototype model of the pitfall trap was constructed by soldering the metal lid to a 340 ml glass jar, 8 cm deep with a 28.5 cm inner diameter. A hole, 6.5 cm in diameter, was punched in the lid before soldering. A similar-sized hole was cut out of the bottom of the pail and covered with a piece of perforated brass sheeting of the kind used for insertion-type traps. The brass was soldered to the floor of the pail around the periphery of the hole. The jar could be screwed into the lid from below the pail. This trap detects more insects than insertion-type traps immersed in the seed bulk. Reed et al. (1991) reported that the early detection of insect populations in seed is best accomplished by pitfall traps.
A Berlese funnel consists of a metal funnel screened at the bottom. It can contain a seed sample of about 1 kg. Heat, usually from a 60 W electric light bulb placed above the seed surface, drives insects and mites down through the screened bottom. The funnel is placed over a jar containing about 50 ml water or 70% alcohol to kill and preserve the insects and mites. The liquid can be studied under a microscope to identify and count the insects and mites that were in the grain sample (Muir, 1997).
Hidden insect infestation can be detected with ultrasonic signals. The feeding activity of insects is monitored using ultrasonic emissions at a particular frequency. Ultrasonic emissions can be produced by feeding, but not by movement, from early in the first instar to the last instar. The number of feeding events is typically proportional to the number of insects per seed. Rice weevils and moths can be detected using this technique.
A portable device has been developed at a stored-products entomology and acarology laboratory in Bordeaux, France. The device is used to monitor the typical signals produced by insect activity. This device accurately monitors the insect infestation in a bin without sampling. Insect presence is detected long before the infestation becomes a risk for long-term storage (Lessard and Andrieu, 1986).
Detection of insects using X-rays has been studied by several researchers, analysing images of the different life stages of insects inside the kernels (Cotton and Wilbur, 1982; Pederson, 1992; Karunakaran et al., 2003). At an experimental level, >97% accuracy can be achieved in detecting the larvae and pupae of rice weevils with X-ray imaging (Karunakaran et al., 2003).
Infested seeds can be separated from sound ones by placing seed samples in a liquid solution with a specific gravity. This allows uninfested seeds to sink, while infested ones float.
Cracking and flotation are official methods of the Association of Official Analytical Chemists (methods 44.041 and 44.042, respectively; AOAC, 1984). Insect materials are separated and floated to the surface of a solution. The floated materials are collected on filter paper and examined microscopically (Cotton and Wilbur, 1982).
Because uric acid is an important constituent of insect excreta, the measurement of uric acid content in stored seeds can correlate to the extent of insect infestation (Subrahmanyan et al., 1955). However, the uric acid could be from an old infestation rather than a current one.
As insects develop in seeds, they respire and produce CO2 as a by-product of their metabolism (Pederson, 1992). Howe and Oxley (1952) used a simple gas analyser and determined that 1% CO2 produced in a standard sample over a 24-h period in a sealed container was indicative of approximately 25 larvae in 450 g seed. An infrared CO2 analyser is more sensitive and quicker for the routine inspection of hidden insect infestations after harvesting (Zisman and Calderon, 1990). Infrared gas analysers can detect 0.15-0.3% CO2 developed by 1-2 insects in a kilogram of seeds within 48 h. Infrared gas analysers can also be used to determine the intergranular CO2 content by sampling from one or more points in a seed bulk.
Nuclear magnetic resonance spectroscopy is a non-destructive method to determine insect infestation in seeds. It gives the images of peaks coming from water and lipids in larvae, which can be correlated with weevil development and seed kernel weight loss (Pederson, 1992).
Dennis and Decker (1962) used ninhydrin-treated papers to determine insect infestation in seeds. Free amino acids in the body fluids of insects react with ninhydrin and produce purple spots on the paper (Pederson, 1992).
The 'insect-detect' immunoassay has been reported to provide the most accurate measurement of actual insect infestation when compared to three traditional methods - X-ray analysis, cracking and flotation, and the insect fragment test (Brader et al., 2002).
NIR spectroscopy is a procedure that can rapidly detect and measure the chemical composition of biological materials. When the wavelength of the incident infrared energy corresponds to the frequency of vibration of a specific molecule, this energy is absorbed by the molecule. Optical sensors measure this absorption and the amount can be related to the concentration of a particular constituent. Dowell et al. (1998) used NIR spectroscopy (1000-1660 nm) to detect infestation of weevils and moths.
Insect infestation can be detected by a locomotor test. Different species respond to y-irradiation in different ways and their locomotor activity and/ or ability to disperse is highly affected. The locomotor activity of y-irradiated beetles in stored products is inversely proportional to the dose applied (Ignatowicz et al., 1994). A lethal dose of 0.3-1.0 kGy for radiation disinfes-tation has been suggested by Ignatowicz et al. (1994) to lower the locomotor activity of confused flour beetles.
Different stains are used to detect weevil infestations (eggs, larvae, pupae or adults) in seeds. Weevils chew a small hole through the seed coat into the endosperm, in which an egg is deposited. As the ovipositor is withdrawn, the female secrets a gelatinous plug that fills the egg channel so that the egg cavity is difficult to detect without a microscope. Various stains have been discovered that will colour the egg plug without staining the seed coat, unless it has been damaged mechanically (Cotton and Wilbur, 1982). Wongo (1990) found that a water-soluble fluorescent dye, berberine sulphate, can stain egg plugs yellow.
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