Yonglin Ren

Proteomic evaluation of adults of Rhyzopertha dominica resistant to phosphine

A proteomic study using a PH(3)-susceptible (RD2) and -resistant strain (CRD343) of Rhyzopertha dominica was undertaken to validate the relation between change of proteins and PH(3) resistance. Protein expression levels were compared using PD-Quest program after two-dimensional polyacrylamide gel-electrophoresis. Comparing the intensity of proteins, 15 proteins decreased and 6 proteins were newly expressed in CRD343. After MALDI-TOF and LC-MS/MS analyses, the decreased proteins were identified as arginine kinases, dihydrolipoamide dehydrogenase, Hsp60, reverse transcriptase, glyceraldehydes-3-phosphate dehydrogenase, triosephosphate isomerase and hypothetical proteins. Dihydrolipoamide dehydrogenase is involved in the Krebs cycle and glyceraldehydes-3-phosphate dehydrogenase and triosephosphate isomerase are involved in the glycolysis pathway. Among up-regulated proteins, sodium channel, glutamate racemase, enolase and vitellogenin were identified. Taken together, PH(3) affected glycolysis as well as Krebs cycle and the induction of enolase might recover this dysfunction.

Effect of ozone on respiration of adult Sitophilus oryzae (L.), Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.).

The effect of ozone on the respiration of three species of adult stored-product Coleoptera was tested in an air-tight flask. Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) adults were exposed to atmosphere containing 0.1, 0.2 or 0.4microg/ml initial ozone at 23-25 degrees C and 50% r.h. Carbon dioxide (CO(2)) production reflected the respiration rates of insects and was determined with a gas chromatograph (GC). The experiments showed that the effects of ozone on respiration had two distinct phases. Phase 1 involved a lower respiration rate of the adult stored-product Coleoptera under ozone atmosphere and reflected the need for insects to reduce ozone toxicity. After 1h, CO(2) production of S. oryzae was 3.19, 2.63, 2.27 and 1.99microl/mg for the ozone concentration of 0, 0.1, 0.2 and 0.4microg/ml, respectively. The results also showed that there were decreases in the rate of respiration in R. dominica and T. castaneum with an increase in ozone concentration. During phase 2, respiration of S. oryzae, R. dominica, and T. castaneum adults treated with ozone increased as the ozone degraded to oxygen. After 7h, the effect of ozone on CO(2) production, relative to the control, changed from a decrease to an increase. The findings in relation to control strategies were discussed.

Toxicity of Ethanedinitrile to Anoplophora glabripennis (Coleoptera: Cerambycidae) Larvae

The mortality of naked Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae) larvae exposed to ethanedinitrile (cyanogen; C2N2) varied with temperature, time of exposure, and dose of ethanedinitrile. The concentration x time (Ct) product of ethanedinitrile over a range of temperatures (4.4, 10.1, 15.6, and 20.1 degrees C) decreased with increasing temperature, for both 3- and 6-h exposures. The Ct products varied with time of exposure at different temperatures. The variations in mortality at different temperatures are described with a slope ratio (Y = [slope]6h/ [slope]3h). At different temperatures, the concentration of ethanedinitrile and the duration of exposure play different roles in killing A. glabripennis larvae. These results suggest the control of A. glabripennis larvae within wood is achievable.

Improved methodology for studying diffusion, sorption and desorption in timber fumigation

Methods to study movement of fumigant through timber were improved, and procedures were developed that obviated the need for sampling lines. Movement for two fumigants (methyl bromide and carbonyl sulphide) through, and sorption on, heartwood of softwood and hardwood were studied. Each fumigant was sorbed less on softwood than on hardwood and penetrated softwood better than hardwood. Neither fumigant penetrated across the grain. Carbonyl sulphide penetrated timber better than did methyl bromide, and was less sorbed on timber. A rapid method of solvent extraction was developed to enable rapid estimation of the amount of intact fumigant sorbed in wood. This procedure enabled excellent recovery of methyl bromide as either intact fumigant or as bromide ion.

Fumigation of Wheat Using Liquid Ethyl Formate Plus Methyl Isothiocyanate in 50-Tonne Farm Bins

Abstract Australian Standard White wheat, Triticum aestivum L. (a marketing grade with mixed grain hardness), with a moisture content of 12.5% was fumigated with a new ethyl formate formulation (95% ethyl formate plus 5% methyl isothiocyanate) identified and developed by Commonwealth Scientific and Industrial Research Organization Entomology, Canberra, Australia. Wheat was fumigated with the formulation at a calculated application rate of 80 g/m3 in two 50-tonne sealed metal vertical silos located at Fisherman Islands, Queensland, Australia. Access was gained through the top of the silo where the application of the formulation was completed within a few minutes by pouring it onto the top of the wheat. After 2 h of recirculation, using a 0.5-kW fan, the in-bin concentrations of ethyl formate achieved equilibrium with a concentration variation <7%. The ethyl formate concentration, in both silos 1 and 2, during the first day’s exposure period remained above 10 g/m3. The concentration of ethyl formate by time product achieved was 790 and 650 g h/m3 in silos 1 and 2, respectively. In silo 1, the formulation was sufficient to kill all life stages of mixed age cultures of Sitophilus oryzae (L.), Rhyzopertha dominica (F.), and Tribolium castaneum (Herbst). In silo 2, control was 100% for R. dominica and T. castaneum and 99.4% for S. oryzae. After 5 d fumigation, the silo top-hatch was opened but no forced aeration was initiated. The in-bin concentration of ethyl formate was lower than the Australian experimental threshold limit value of 100 ppm. The ethyl formate and methyl isothiocyanate residues in the grain had declined to below the Australian experimental maximum residue limit of 0.2 and 0.1 mg/kg, respectively. The workspace and environmental levels of ethyl formate and methyl isothiocyanate were less than the detection limit of 0.1 ppm. The treatment with ethyl formate formulation had no affect on the wheat germination and seed color compared with untreated controls.

Natural levels of ethyl formate in stored grains determined using an improved method of analysis

Ethyl formate was readily determined with a gas chromatograph (GC) equipped with a flame ionisation detector. Natural levels of ethyl formate in Australian wheat, barley, oats, and canola were analysed by GC, after extraction with ammonium nitrate solution. Background levels of ethyl formate were present in newly harvested and stored grain. The levels of ethyl formate (0.1-0.6 mg kg-1) in grains varied with commodity, temperature, moisture and period of storage. The values ranged from 0.1 to 0.2 mg kg -1 for newly harvested wheat, barley and oats, and 0.3-0.4 mg kg -1 for newly harvested canola. Ethyl formate was present in grains at harvest, increased during the first 7 months of storage, and then began to decline, particularly at grain temperatures higher than 25°C and moisture contents higher than 12.5% (for wheat, barley and oats) and 6.5% (for canola). The natural levels of ethyl formate should be considered when establishing maximum residue limits.

Ethyl formate plus methyl isothiocyanate--a potential liquid fumigant for stored grains.

BACKGROUND Methyl bromide is being phased out for use on stored commodities, as it is listed as an ozone-depleting substance, and phosphine is the fumigant widely used on grains. However, phosphine resistance occurs worldwide, and phosphine fumigation requires a long exposure period and temperatures of >15 °C. There is an urgent requirement for the development of a fumigant that kills insects quickly and for phosphine resistance management. This paper reports on a new fumigant formulation of 95% ethyl formate plus 5% methyl isothiocyanate as an alternative fumigant for stored grains. RESULTS The formulation is stable for at least 4 months of storage at 45 °C. A laboratory bioassay with the formulation showed that it controlled all stages of Sitophilus oryzae (L.), Sitophilus granarius (L.), Tribolium castaneum (Herbst), Rhyzopertha dominica (F.), Trogoderma variabile Ballion and Callosobruchus maculatus (Fabricius) in infested wheat, barley, oats and peas at 80 mg L(-1) for 5 days, and in canola at both 40 mg L(-1) for 5 days and 80 mg L(-1) for 2 days at 25±2 °C. After an 8-14 day holding period, residues of ethyl formate and methyl isothiocyanate in wheat, barley, peas and canola were below the experimental permit levels of 1.0 and 0.1 mg kg(-1). However, fumigated oats needed an 18 day holding period. CONCLUSIONS The findings suggest that the ethyl formate plus methyl isothiocyanate formulation has potential as a fumigant for the control of stored-grain insect pests in various commodities.

An innovative rapid method for analysis of 10 organophosphorus pesticide residues in wheat by HS-SPME-GC-FPD/MSD

The rapid detection of pesticide residues in wheat has become a top food security priority. A solvent-free headspace solid-phase microextraction (HS-SPME) has been evaluated for rapid screening of organophosphorus pesticide (OPP) residues in wheat with high sensitivity. Individual wheat samples (1.7 g), spiked with 10 OPPs, were placed in a 4 mL sealed amber glass vial and heated at 60°C for 45 min. During this time, the OPP residues were extracted with a 50 μm/30 μm divinylbenzene (DVB)/carboxen (CAR)/plasma desorption mass spectroscopy polydimethylsiloxane (PDMS) fiber from the headspace above the sample. The fiber was then removed and injected into the GC injection port at 250°C for desorption of the extracted chemicals. The multiple residues were identified by a GC mass spectrometer detector (GC-MSD) and quantified with a GC flame photometric detector (GC-FPD). Seven spiked levels of 10 OPPs on wheat were analyzed. The GC responses for a 50 μm/30 μm DVB/CAR/PDMS fiber increased with increasing spiking levels, yielding significant (R(2) > 0.98) linear regressions. The lowest LODs of the multiple pesticide standards were evaluated under the conditions of the validation study in a range of levels from 0 (control) to 100 ng of pesticide residue per g of wheat that separated on a low-polar GC capillary column (Agilent DB-35UI). The results of the HS-SPME method were compared with the QuEChERS AOAC 2007.01 method and they showed several advantages over the latter. These included improved sensitivity, selectivity, and simplicity.

Fungal contaminants of stored wheat vary between Australian states

A survey was undertaken to determine the mycoflora associated with stored wheat in four states across Australia including Western Australia, South Australia, Victoria and New South Wales. A total of 482 fungal isolates from 15 genera were isolated. The most common genera isolated were Alternaria spp., Aspergillus sp., Aureobasidium sp., Cladosporium spp., Drechslera sp., Fusarium spp., Mucor sp., Nigrospora sp., Penicillium sp., Rhizopus sp., Stemphylium sp., Eutiarosporella spp., Ulocladium sp., Epicoccum sp., and an undescribed genus from the Hypocreales. Diversity profiling also identified fungi that were not isolated using traditional methods including Paecilomyces spp., Dendryphiella and Cryptococcus. The results indicate that the mycoflora of stored wheat varies between Australian grain growing regions, and that diversity profiling analysis identifies different fungal contaminants compared to traditional methods. This study also revealed that some pathogens of high risk to humans (e.g. Cryptococcus) may occur with a high frequency in wheat.

Optimization of Headspace Solid-Phase Microextraction Conditions for the Identification of Phytophthora cinnamomi Rands

A robust technique was developed to identify Phytophthora cinnamomi using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography (GC) coupled to a flame ionization detector (FID) for analyzing volatile organic compounds (VOCs). Six fiber types were evaluated and results indicated that the three-phase fiber 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) had the highest extraction efficiency for both polar and nonpolar GC columns. The maximum extraction efficiency (equilibrium absorption) was achieved 16 h after fiber exposure in the HS. Absorbed compounds on the fiber were completely desorbed in the GC injector after 5 min at 250°C. Compared with the nonpolar column, the polar column showed optimum separation of VOCs released from P. cinnamomi. Under the optimized HS-SPME and GC/FID conditions, lower detection limits for the four external standards was found to be between 1.57 to 27.36 ng/liter. Relative standard deviations <9.010% showed that the method is precise and reliable. The method also showed good linearity for the concentration range that was analyzed using four standards, with regression coefficients between 0.989 and 0.995, and the sensitivity of the method was 104 times greater than that of the conventional HS method. In this study, the VOC profiles of six Phytophthora spp. and one Pythium sp. were characterized by the optimized HS-SPME-GC method. The combination of the VOCs creates a unique pattern for each pathogen; the chromatograms of different isolates of P. cinnamomi were the same and the specific VOC pattern of P. cinnamomi remained consistently independent of the growth medium used. The chromatograms and morphological studies showed that P. cinnamomi released specific VOCs at different stages of colony development. Using the optimized HS-SPME GC method, identification of P. cinnamomi from 15 in vivo diseased soil samples was as high as 100%. Results from this study demonstrate the feasibility of this method for identifying P. cinnamomi and the potential use of this method for physiological studies on P. cinnamomi.