Sulfuryl fluoride as a quarantine treatment for Anoplophora glabripennis (Coleoptera: Cerambycidae) in regulated wood packing material

The Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), was probably introduced into the United States from China with solid wood packing and dunnage during the 1980s, and it has recently become established in limited infestations near several major cities in the United States. Regulated wood packing material (RWPM) arriving in the United States from China is required to undergo fumigation with methyl bromide (MeBr), to be heat treated, or kiln dried. Sulfuryl fluoride (SF) is a candidate fumigant to replace MeBr under certain conditions. SF fumigations were conducted in 432-liter Lexan chambers held in a 6.1-m (20-foot) refrigerated container for temperature control. Each fumigation consisted of 12 Populus spp. 10- by 10- by 115-cm timbers, of high moisture content, naturally infested with Asian longhorned beetle. During 2001, we fumigated wood for 24 h at a range of doses (20-112 g/m3) and temperatures (4.4, 10.0, 15.6, and 21.1 degrees C) and subjected the data to probit analysis. Confirmatory fumigations were conducted at doses of 120 and 104 g/m3 at temperatures of 10.0 and 15.6 or 21.1 degrees C, respectively, which resulted in complete kill of all larvae. Pupae that became available later in the year as temperatures warmed were fumigated at 15.6 and 21.1 degrees C with 104 g/m3, which resulted in complete pupal mortality. The next year (2002), we conducted 24-h fumigations with doses of 116 g/m3 at 4.4 and 10.0 degrees C with cold-harvested wood infested with cold-acclimated larvae. Cold-acclimated larvae required much higher concentration times time (CxT) product for control at 4.4 and 10.0 degrees C compared with nonacclimated larvae. Sulfuryl fluoride treatments at a dose of 104 g/m3 and temperature of 15.6 degrees C and above and that achieved a CxT product of 1,095 g-h/m3 or above are recommended for RWPM infested with Asian longhorned beetle larvae and pupae.     

Methyl bromide as a quarantine treatment for Anoplophora glabripennis (Coleoptera: Cerambycidae) in regulated wood packing material

Anoplophora glabripennis (Motschulsky), has recently (since 1996) established in limited infestations near several cities in the United States. A. glabripennis was probably introduced into the United States with solid wood packing or dunnage. During 2001, we evaluated the current APHIS Schedule T404-b-1-1 for methyl bromide (MeBr) fumigation. Fumigations were conducted in 432-liter Lexan chambers inside a 6.1-m refrigerated container. Each fumigation consisted of 12 high-moisture, naturally infested Populus spp. timbers. We fumigated wood for 24 h at 4.4 degrees C (80 g/m3), 10.0 degrees C (64 g/m3), 15.6 degrees C (56 g/m3), and 21.1 degrees C (48 g/m3). All schedule doses resulted in 100% kill of A. glabripennis larvae. During 2002, we conducted additional fumigations to determine the basic toxicity of MeBr to A. glabripennis larvae in solid wood timbers of 10 by 10 by 115-cm size. Probit analysis estimated the CxT product at 99.0, 99.9, 99.99, and 99.99683% kill (probit-9). The probit-9 values for CxT were 1,196.1, 918.7, 642.4, and 362.4 g-h/m3 at 4.4, 10.0, 15.6, and 21.1 degrees C, respectively. Applied doses to achieve this level of control were estimated to be 119.6, 82.7, 56.0, and 32.2 g/m3, respectively. These applied doses are satisfactory for wood as a commodity with wood load factors of approximately 25% and may be higher than necessary for container fumigation where sorptive wood load as crating or pallets may only be 5% or less. The APHIS Schedule T404-b-1-1 is adequate if extended for 24 h and should be amended to include intermediate doses at 10.0 and 15.6 degrees C, thus reducing the use of MeBr at these temperatures.     

Container fumigation as a quarantine treatment for Anoplophora glabripennis (Coleoptera: Cerambycidae) in regulated wood packing material

Anoplophora glabripennis Motschulsky, an Asian cerambicid beetle, was first found in the United States in 1996 and was likely introduced into the United States through infested wood packing materials and dunnage. Methyl bromide (MeBr) fumigation is an internationally accepted treatment for such wood-boring beetles, but the use of MeBr is scheduled for reduction or elimination as alternatives become available. The use of MeBr can be reduced by more efficient and technically sound fumigation techniques, including good circulation and proper loading. Concentration x time products (CxT) reported for A. glabripennis in solid wood timbers were used to test estimated doses in container fumigations conducted in Tianjin and Shanghai, China, during years 2002-2003. We found that CxT products observed were generally adequate and would support reduced doses of MeBr at temperatures intermediate (10.0 and 15.6 degrees C) to the Animal and Plant Health Inspection Service schedule of 80 g and 48 g/m3 at > or =4.4 and > or = 21.1degrees C, respectively. The use of fans is recommended, and loading of the containers should never reach 100% to allow for better circulation and desorption. Proper use of fans will allow for better aeration and increase safety to inspectors and consignees of containerized fumigated commodities.     

Bacterial Oxidation of Methyl Bromide in Fumigated Agricultural Soils

The oxidation of [(sup14)C]methyl bromide ([(sup14)C]MeBr) to (sup14)CO(inf2) was measured in field experiments with soils collected from two strawberry plots fumigated with mixtures of MeBr and chloropicrin (CCl(inf3)NO(inf2)). Although these fumigants are considered potent biocides, we found that the highest rates of MeBr oxidation occurred 1 to 2 days after injection when the fields were tarped, rather than before or several days after injection. No oxidation of MeBr occurred in heat-killed soils, indicating that microbes were the causative agents of the oxidation. Degradation of MeBr by chemical and/or biological processes accounted for 20 to 50% of the loss of MeBr during fumigation, with evasion to the atmosphere inferred to comprise the remainder. In laboratory incubations, complete removal of [(sup14)C]MeBr occurred within a few days, with 47 to 67% of the added MeBr oxidized to (sup14)CO(inf2) and the remainder of counts associated with the solid phase. Chloropicrin inhibited the oxidation of MeBr, implying that use of this substance constrains the extent of microbial degradation of MeBr during fumigation. Oxidation was by direct bacterial attack of MeBr and not of methanol, a product of the chemical hydrolysis of MeBr. Neither nitrifying nor methane-oxidizing bacteria were sufficiently active in these soils to account for the observed oxidation of MeBr, nor could the microbial degradation of MeBr be linked to cooxidation with exogenously supplied electron donors. However, repeated addition of MeBr to live soils resulted in higher rates of its removal, suggesting that soil bacteria used MeBr as an electron donor for growth. To support this interpretation, we isolated a gram-negative, aerobic bacterium from these soils which grew with MeBr as a sole source of carbon and energy.     

Host range of the emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae) in North America: results of multiple-choice field experiments

Emerald ash borer (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae), an invasive phloem-feeding pest, was identified as the cause of widespread ash (Fraxinus) mortality in southeast Michigan and Windsor, Ontario, Canada, in 2002. A. planipennis reportedly colonizes other genera in its native range in Asia, including Ulmus L., Juglans L., and Pterocarya Kunth. Attacks on nonash species have not been observed in North America to date, but there is concern that other genera could be colonized. From 2003 to 2005, we assessed adult A. planipennis landing rates, oviposition, and larval development on North American ash species and congeners of its reported hosts in Asia in multiple-choice field studies conducted at several southeast Michigan sites. Nonash species evaluated included American elm (U. americana L.), hackberry (Celtis occidentalis L.), black walnut (J. nigra L.), shagbark hickory [Carya ovata (Mill.) K.Koch], and Japanese tree lilac (Syringa reticulata Bl.). In studies with freshly cut logs, adult beetles occasionally landed on nonash logs but generally laid fewer eggs than on ash logs. Larvae fed and developed normally on ash logs, which were often heavily infested. No larvae were able to survive, grow, or develop on any nonash logs, although failed first-instar galleries occurred on some walnut logs. High densities of larvae developed on live green ash and white ash nursery trees, but there was no evidence of larval survival or development on Japanese tree lilac and black walnut trees in the same plantation. We felled, debarked, and intensively examined >28 m2 of phloem area on nine American elm trees growing in contact with or adjacent to heavily infested ash trees. We found no sign of A. planipennis feeding on any elm.     

Evaluation of double-decker traps for emerald ash borer (Coleoptera: Buprestidae)

Improved detection tools are needed for the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), an invasive forest insect from Asia that has killed millions of ash (Fraxinus spp.) trees in North America since its discovery in Michigan in 2002. We evaluated attraction of adult A. planipennis to artificial traps incorporating visual (e.g., height, color, silhouette) and olfactory cues (e.g., host volatiles) at field sites in Michigan. We developed a double-decker trap consisting of a 3-m-tall polyvinyl pipe with two purple prisms attached near the top. In 2006, we compared A. planipennis attraction to double-decker traps baited with various combinations of manuka oil (containing sesquiterpenes present in ash bark), a blend of four ash leaf volatiles (leaf blend), and a rough texture to simulate bark. Significantly more A. planipennis were captured per trap when traps without the rough texture were baited with the leaf blend and manuka oil lures than on traps with texture and manuka oil but no leaf blend. In 2007, we also tested single prism traps set 1.5 m above ground and tower traps, similar to double-decker traps but 6 m tall. Double-decker traps baited with the leaf blend and manuka oil, with or without the addition of ash leaf and bark extracts, captured significantly more A. planipennis than similarly baited single prism traps, tower traps, or unbaited double-decker traps. A baited double-decker trap captured A. planipennis at a field site that was not previously known to be infested, representing the first detection event using artificial traps and lures. In 2008, we compared purple or green double-decker traps, single prisms suspended 3-5 m above ground in the ash canopy (canopy traps), and large flat purple traps (billboard traps). Significantly more A. planipennis were captured in purple versus green traps, baited traps versus unbaited traps, and double-decker versus canopy traps, whereas billboard traps were intermediate. At sites with very low A. planipennis densities, more A. planipennis were captured on baited double-decker traps than on other traps and a higher percentage of the baited double-decker traps captured beetles than any other trap design. In all 3 yr, peak A. planipennis activity occurred during late June to mid-July, corresponding to 800-1200 growing degree-days base 10 degrees C (DD10). Nearly all (95%) beetles were captured by the end of July at approximately 1400 DD10.     

Estimating potential emerald ash borer (Coleoptera: Buprestidae) populations using ash inventory data

Emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), a phloem-feeding pest native to Asia, was identified in June 2002 as the cause of widespread ash (Fraxinus spp.), mortality in southeastern Michigan and Windsor, Ontario, Canada. Localized populations of A. planipennis have since been found across lower Michigan and in areas of Ohio, Indiana, Illinois, Maryland, and Ontario. Officials working to contain A. planipennis and managers of forestlands near A. planipennis infestations must be able to compare alternative strategies to allocate limited funds efficiently and effectively. Empirical data from a total of 148 green ash, Fraxinus pennsylvanica Marsh., and white ash, Fraxinus americana L., trees were used to develop models to estimate surface area of the trunk and branches by using tree diameter at breast height (dbh). Data collected from 71 additional F. pennsylvanica and F. americana trees killed by A. planipennis showed that on average, 88.9 +/- 4.6 beetles developed and emerged per m2 of surface area. Models were applied to ash inventory data collected at two outlier sites to estimate potential production of A. planipennis beetles at each site. Large trees of merchantable size (dbh > or = 26 cm) accounted for roughly 6% of all ash trees at the two sites, but they could have contributed 55-65% of the total A. planipennis production at both sites. In contrast, 75- 80% of the ash trees at the outlier sites were < or =13 cm dbh, but these small trees could have contributed only < or =12% of the potential A. planipennis production at both sites. Our results, in combination with inventory data, can be used by regulatory officials and resource managers to estimate potential A. planipennis production and to compare options for reducing A. planipennis density and slowing the rate of spread for any area of interest.     

Oxidation of methyl halides by the facultative methylotroph strain IMB-1.

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [(14)C]formaldehyde to (14)CO(2) but had only a small capacity for oxidation of [(14)C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [(14)C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2). The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

Effects of chipping, grinding, and heat on survival of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae), in chips

The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), a phloem-feeding insect from Asia, was identified in 2002 as the cause of widespread ash (Fraxinus sp.) mortality in southeastern Michigan and Essex County, Ontario. Most larvae overwinter as nonfeeding prepupae in the outer sapwood or thick bark of large trees. In a series of studies, we evaluated effects of grinding, chipping, and heat treatment on survival of A. planipennis prepupae in ash material. Heavily infested ash bolts containing roughly 8,700 prepupae were processed by a horizontal grinder with either a 2.5- or 10-cm screen. There was no evidence of A. planipennis survival in chips processed with the 2.5-cm screen, but eight viable prepupae were recovered from chips processed with the 10-cm screen. We chiseled additional sentinel chips with prepupae from ash logs and buried 45 in each chip pile. In total, six prepupae in sentinel chips survived the winter, but we found no sign of adult A. planipennis emergence from the processed chips. Subsequently, we assessed prepupal survival in chips processed by a chipper or a horizontal grinder fit with 5-, 10-, or 12.7-cm screens. An estimated 1,565 A. planipennis prepupae were processed by each treatment. Chips from the chipper were shorter than chips from the grinder regardless of the screen size used. No live prepupae were found in chips produced by the chipper, but 21 viable prepupae were found in chips from the grinder. Infested wood and bark chips chiseled from logs were held in ovens at 25, 40, or 60 degrees C for 8, 24, or 48 h. Prepupal survival was consistently higher in wood chips than bark chips at 40 degrees C, whereas no prepupae survived exposure to 60 degrees C for eight or more hours. In a second study, prepupae in wood chips were exposed to 40, 45, 50, 55, or 60 degrees C for 20 or 120 min. Some prepupae survived 20 min of exposure to all temperatures. No prepupae survived exposure to 60 degrees C for 120 min, but 17% survived exposure to 55 degrees C for 120 min, suggesting that some fraction of the population may survive internationally recognized phytosanitary standards (ISPM-15) for treatment of wood packing material.     

Effects of cutting date, outdoor storage conditions, and splitting on survival of Agrilus planipennis (Coleoptera: Buprestidae) in firewood logs

The emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) is an exotic pest of ash (Fraxinus spp.) in North America. We conducted studies in Michigan to evaluate how different tree cutting dates, outdoor storage conditions, and splitting affected A. planipennis survival in firewood logs. In 2002-2003, we cut logs from A. planipennis-infested ash trees each month, from July to October, and stored half of the logs in shade and half in full sun. In 2003-2004, we tested logs cut July-December; stored in sun versus shade; tarped versus untarped; and whole logs versus split logs. For both years, A. planipennis successfully emerged the summer after cutting from logs that represented all treatments and all cutting dates tested. Adult emergence density was significantly lower in logs cut in July and August. In 2003-2004, A. planipennis adult length was significantly shorter, and percentage of mortality was significantly higher for logs cut in August compared with later months. Emergence density was significantly lower for split logs compared with whole logs for all cutting months except for December. There was no significant difference in adult emergence density between logs stored in full sun versus shade in 2002-2003. In 2003-2004, untarped logs in full sun or shade had significantly lower adult emergence densities than tarped logs in the sun or shade. In conclusion, emergence, survival, and size of A. planipennis was significantly reduced if logs were cut early during larval development (July or August); splitting logs and storing them untarped in full sun or shade further reduced adult emergence. No treatment was 100% effective in preventing adult emergence.     

Impact of fumigants on soil microbial communities.

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.     

Temperature responses of carbon dioxide fluxes from coarse dead wood in a black ash wetland

Wetlands Ecology and Management - The invasive emerald ash borer (EAB, Agrilus planipennis Fairmaire) causes widespread ash tree mortality in North America, and the CO2 efflux (respiration, F) from...     

The Destruction of Byssochlamys fulva asci by Low Concentrations of Gaseous Methyl Bromide and by Aqueous Solutions of Chlorine, an Iodophor and Peracetic Acid

S ummary : Byssochlamys fulva asci are resistant to high concentrations of aqueous chlorine and iodophor solutions, but are sensitive to peracetic acid. Concentrations of 2% and 4% of peracetic acid gave 99·9% reductions in 2·5 and 1·3 min, respectively. The asci were also sensitive to methyl bromide gas (MeBr), c. 5 × 10⁴ asci/g inoculated into tapioca starch powder ( a _w= 0·69) being killed in 30 days by 90 mg of MeBr/kg of starch. About 180 asci/g were killed by adding 60 mg of MeBr/kg of starch.     

Effects of trap type, placement and ash distribution on emerald ash borer captures in a low density site

Effective methods for early detection of newly established, low density emerald ash borer (Agrilus planipennis Fairmaire) infestations are critically needed in North America. We assessed adult A. planipennis captures on four types of traps in a 16-ha site in central Michigan. The site was divided into 16 blocks, each comprised of four 50- by 50-m cells. Green ash trees (Fraxinus pennsylvanica Marshall) were inventoried by diameter class and ash phloem area was estimated for each cell. One trap type was randomly assigned to each cell in each block. Because initial sampling showed that A. planipennis density was extremely low, infested ash logs were introduced into the center of the site. In total, 87 beetles were captured during the summer. Purple double-decker traps baited with a blend of ash leaf volatiles, Manuka oil, and ethanol captured 65% of all A. planipennis beetles. Similarly baited, green double-decker traps captured 18% of the beetles, whereas sticky bands on girdled trees captured 11% of the beetles. Purple traps baited with Manuka oil and suspended in the canopies of live ash trees captured only 5% of the beetles. At least one beetle was captured on 81% of the purple double-decker traps, 56% of the green double-decker traps, 42% of sticky bands, and 25% of the canopy traps. Abundance of ash phloem near traps had no effect on captures and trap location and sun exposure had only weak effects on captures. Twelve girdled and 29 nongirdled trees were felled and sampled in winter. Current-year larvae were present in 100% of the girdled trees and 72% of the nongirdled trees, but larval density was five times higher on girdled than nongirdled trees.     

Oxidation of Methyl Halides by the Facultative Methylotroph Strain IMB-1

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 μM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [¹⁴C]formaldehyde to ¹⁴CO₂ but had only a small capacity for oxidation of [¹⁴C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [¹⁴C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K_s values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO₂. The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

Na2CO3 influence on DNA double helix stability: strong anion destabilizing effect

Addition of Na(2)CO(3) to almost salt-free DNA solution (5.10(-5)M EDTA, pH=5.7, T(m)=26.5 degrees C) elevates both pH and the DNA melting temperature (T(m)) if Na(2)CO(3) concentration is less than 0.004 M. For 0.004 M Na(2)CO(3), T(m)=58 degrees C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in T(m) to 37 degrees C for 1M Na(2)CO(3) (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04 M Na(2)CO(3) (T(m)=48.3 degrees C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56/10.87) and a wide Na(2)CO(3) concentration interval (0.004/1M), a procedure has been developed for determining the separate influences on T(m) of Na(+), pH, and anions formed by Na(2)CO(3) (HCO(3)(-) and CO(3)(2-)). Comparison of influence of anions formed by Na(2)CO(3) on DNA stability with Cl(-) (anion inert to DNA stability), ClO(4)(-) (strong DNA destabilizing "chaotropic" anion) and OH(-) has been carried out. It has been shown that only Na(+) and pH influence T(m) in Na(2)CO(3) solution at concentrations lower than 0.001 M. However, the T(m) decrease with concentration for [Na(2)CO(3)]>/=0.004 M is only partly caused by high pH=10.7. Na(2)CO(3) anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na(2)CO(3) (pH=10.84, [Na(+)]=0.2M, T(m)=42.7 degrees C), the anion destabilizing effect is higher 20 degrees C. For NaClO(4) (ClO(4)(-) is a strong "chaotropic" anion), an equal anion effect occurs at much higher concentrations approximately 3M. This means that Na(2)CO(3) gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na(2)CO(3) as well as after addition of NaOH for pH greater than 11.2.     

Reductive nitrosylation and peroxynitrite-mediated oxidation of heme-hemopexin

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2).     

Temperature response of mesophyll conductance. Implications for the determination of Rubisco enzyme kinetics and for limitations to photosynthesis in vivo

CO(2) transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g(m)), is finite. Therefore, it will limit photosynthesis when CO(2) is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude of g(m). The process dominating g(m) is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO(2) in water have all been suggested. The response of g(m) to temperature (10 degrees C-40 degrees C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q(10)) of approximately 2.2 for g(m), suggesting control by a protein-facilitated process because the Q(10) for diffusion of CO(2) in water is about 1.25. Further, g(m) values are maximal at 35 degrees C to 37.5 degrees C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g(m) to calculate CO(2) at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10 degrees C to 40 degrees C. Using these parameters, we determined the limitation imposed on photosynthesis by g(m). Despite an exponential rise with temperature, g(m) does not keep pace with increased capacity for CO(2) uptake at the site of Rubisco. The fraction of the total limitations to CO(2) uptake within the leaf attributable to g(m) rose from 0.10 at 10 degrees C to 0.22 at 40 degrees C. This shows that transfer of CO(2) from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.     

Methyl bromide fumigation for the control of Aspergilli and Penicillia in stored grains

Fumigation with methyl bromide (MeBr) at a concentration of 120 mg/1 maintained for 4 h at 25°C caused 100% mortality of spores of Aspergillus ochraceus, A. flavus, Penicillium citrinum, P. chrysogenum and P. cyclopium. However, 40% of an A. niger spore population retained its viability after this treatment. Increasing the duration of fumigation to 24 h at a concentration of 40 mg/1 MeBr caused 100% spore mortality of all fungi tested. Total growth inhibition of 24 h-old mycelia was achieved with 40 mg/1 for 24 h or 120 mg/1 for 4 h. These concentrations for the same period of exposure were not inhibitory for 7-day-old mycelia of any of the fungi tested. In A. niger-inoculated wheat grains fumigated with 100 mg/1 MeBr for 24 h, 20% yielded fungal contaminants after 16 days of storage and 100% after 29 days. There was a marked drop in the percent germination of the grains after fumigation, whereas the free fatty acids level was higher than in unfumigated grain. The results of the in vivo study suggest that MeBr given at a commercial dosage for 24 h is not only ineffective in destroying the internal inocula of wheat grains but also enables their subsequent development by weakening the resistance of grains to fungal attack.     

Degradation of methyl bromide by methanotrophic bacteria in cell suspensions and soils.

Cell suspensions of Methylococcus capsulatus mineralized methyl bromide (MeBr), as evidence by its removal from the gas phase, the quantitative recovery of Br- in the spent medium, and the production of 14CO2 from [14C]MeBr. Methyl fluoride fluoride (MeF) inhibited oxidation of methane as well as that of [14C]MeBr. The rate of MeBr consumption by cells varied inversely with the supply of methane, which suggested a competitive relationship between these two substrates. However, MeBr did not support growth of the methanotroph. In soils exposed to high levels (10,000 ppm) of MeBr, methane oxidation was completely inhibited. At this concentration, MeBr removal rates were equivalent in killed and live controls, which indicated a chemical rather than biological removal reaction. At lower concentration (1,000 ppm) of MeBr, methanotrophs were active and MeBr consumption rates were 10-fold higher in live controls than in killed controls. Soils exposed to trace levels (10 ppm) of MeBr demonstrated complete consumption within 5 h of incubation, while controls inhibited with MeF or incubated without O2 had 50% lower removal rates. Aerobic soils oxidized [14C]MeBr to 14CO2, and MeF inhibited oxidation by 72%. Field experiments demonstrated slightly lower MeBr removal rates in chambers containing MeF than in chambers lacking MeF. Collectively, these results show that soil methanotrophic bacteria, as well as other microbes, can degrade MeBr present in the environment.