Enzyme activities can be used as an index of microbial functional diversity, although accumulated enzymes may contribute considerably to the overall enzyme activity of a soil. A semi-quantitative method to determine enzyme protein contents in soil based on the specific activities of reference enzymes and enzyme activity values of soils was reported by Klose and Tabatabai in order to prove whether there is a direct correlation between the activity of any enzyme and its protein concentration in soil. This approach is based on the assumption that the compositions of the reference enzymes are similar to those in soils. Protein concentrations were suggested to serve as a suitable measure to quantify the effects of environmental changes, for example after application of pesticides, on soil biological properties . The understanding of the impacts of pesticide fumigants on key biochemical reactions involved in organic matter degradation and soil nutrient dynamics is important in order to evaluate the ecological significance of fumigation on the soil system. The toxicity of fumigants is related to their interference with respiratory enzymes, including pyruvate dehydrogenase, their ability to chelate metal cations such as Cu, the inhibition by the unchelated ion,blueberry grow bag and toxic degradation products such as methyl isothiocyanate . MeBr can be degraded in soils by the following three pathways : a) chemical hydrolysis to form methanol and bromide, b) methylation to soil organic matter and release of bromide ion, and c) microbial oxidation to form formaldehyde and bromide ion.
Biological hydrolysis and other microbial processes involving enzymatic processes are also likely to contribute to the degradation of MeBr in soil . Microbial respiration, nitrification potential, and dehydrogenase and arylsulfatase activities were inhibited by MeBr + CP and the alternatives PrBr, InLine, Midas and CPEC one week after soil fumigation . After 30 weeks, there was no difference in microbial biomass and activities between the treatments studied, with the exception of lower acid phosphatase and arylsulfatase activities in fumigated soils. These results indicate that there are short- and long-term differences in the response of various microbial and enzymatic processes to MeBr + CP and alternative fumigants and thus, of the various functions of the soil biota in ecosystems. A limitation of this study is that it was conducted for a maximum of 37 weeks; it remains unknown if MeBr + CP and alternative fumigants have longer-term impacts on soil biochemical processes under field conditions after multiple applications. The objective of this study was to evaluate the effect of repeated soil fumigation with MeBr + CP and two registered and two non-registered alternative fumigants on microbial biomass and respiration, the activities of dehydrogenase, acid phosphatase, β-glucosidase and arylsulfatase, and enzyme protein concentrations in soils. Furthermore, the effect of these fumigants was evaluated on dry proteins containing β-glucosidase, acid phosphatase and arylsulfatase in the absence of immobilizing or protecting constituents of soil .
The selected alternative fumigants represent the actual formulations that likely will be used by growers for strawberry production. Dehydrogenase activities were selected because they reflect the total oxidative activities of soil microorganisms and are important in oxidizing soil organic matter. Acid phosphatase catalyzes the hydrolysis of a variety of organic phosphomonoesters and is therefore important in soil organic P mineralization and plant nutrition. The enzyme β-glucosidase catalyzes the hydrolysis of cellobiose, and thus plays a major role in the initial phases of the decomposition of organic C compounds. Arylsulfatase is believed to be partly responsible for S cycling in soils as it participates in the process whereby organic sulfate esters are mineralized and made available for plants. The first aim of the present study was to test whether soil fumigation with these four potential pesticides will alter important soil functions that, in turn, will affect the long-term productivity of agricultural soils. The second aim of this study was to evaluate the effects of soil fumigation on the activities of enzyme proteins, which may be present in the soil as free enzymes and not protected by clay-humus complexes. Free enzymes are likely to be more sensitive to environmental factors as intracellular or adsorbed enzymes, which are protected by the cell envelope or by clay-humic complexes. Field studies were conducted in California, USA, in the central region in Watsonville and in the southern region in Oxnard in 2000 and 2001. Both sites are located in intensive strawberry production areas of California. Soil at both locations had not been fumigated for the past 2 and 3 years prior to this experiment for Watsonville and Oxnard site, respectively. However, before that soil at both sites had been fumigated routinely with MeBr + CP for the past 10 years. The soil in Watsonville is classified as an Elder sandy loam . The soil in Oxnard is classified as a Hueneme sandy loam .
The past 50-year average annual precipitation is 582 mm and 385 mm at Watsonville and Oxnard, respectively. The average annual maximum and minimum temperature at Watsonville is 19.5ºC and 10.7ºC, respectively. Corresponding values for Oxnard are 21.2ºC and 10.7ºC. Commercial agricultural practices for the area were followed . The soil was tilled and beds were formed in Watsonville at 132 cm center-to-center and in Oxnard at 173 cm center-to-center . Slow release fertilizer was applied to the beds at the rate of 400 kg ha-1 y -1. A drip irrigation system was used consisting of two drip tapes , with emitters spaced 30 cm apart and an emitter flow rate of 0.87 l min-1 at 70 kPa, placed 10 cm and 30 cm from the bed center at a soil depth ranging from 2 to 5 cm. Fumigation treatments were randomized in a complete block design with four replicates per treatment at each site. Fumigants used, fumigant rates and application methods are summarized in Table 1. Each replicate consisted of three neighboring 15-m long beds.Soil in Watsonville was fumigated on August 10, 2000 and September 27, 2001, the soil in Oxnard was fumigated on September 1, 2000 and August 24, 2001. At the time of fumigation, the average daily soil temperature within the raised bed ranged between 16 to 20ºC, and the average soil water content was less than 85% of field capacity . About 4 weeks after fumigation bareroot strawberry [Fragaria X ananassa Duchesne, variety “Diamante” and “Camarosa” ] was transplanted in 2000 and 2001. Pesticide effects on soil microorganisms are difficult to evaluate because of the heterogeneous physical-chemical nature of soil, resulting in uncertainties about their distribution and fate within soil microsites. Previous studies on the effects of potential MeBr alternatives on the size,blueberry grow bag size composition and activity of soil microorganisms are limited to one or a few fumigants, a relative short time period, and/or the laboratory . Recovery of microbial processes in the laboratory compared to the field may be reduced due to the absence of re-colonization by nonfumigated soils . Furthermore, the effect of alternative fumigants on soil microbial processes was studied on soils with a 10-year history of fumigation with MeBr + CP combinations followed by a 2 to 3 year break prior to the initiation of these field experiments at Watsonville and Oxnard, respectively. Consequently, results obtained from these soils with a long-term fumigation history may not apply to soils previously not fumigated . The results presented in this work are part of a longer study to evaluate application methods and efficacy of chemical MeBr alternatives to control weeds and pathogens in strawberry production systems in California, USA. The response of microbial performance to soil fumigation with InLine, CP, PrBr and Midas relative to the standard MeBr + CP application and a control soil was determined at 1, 4, and 30 weeks after fumigation in 2000, the first year of the study. Fumigation initially inhibited microbial respiration, nitrification potential, and activities of dehydrogenase, acid phosphatase and arylsulfatase . After 30 weeks, microbial activities in fumigated and control soils were similar at both sites, with exception of acid phosphatase and arylsulfatase activities in selected treatments that remained lower in the fumigated soils. Soil microbial biomass C and β-glucosidase activity were not affected by fumigation with MeBr + CP and alternatives throughout the whole study period in the first year .
This paper focused on the effects of repeated soil fumigation with MeBr + CP, PrBr, InLine, Midas, and CP on the size and activity of soil microorganisms and hydrolytic enzymes, which control the degradation of organic substances and the rate at which nutrient elements become available for plants . Microbial respiration was significantly decreased in Oxnard soils fumigated with MeBr + CP, but not affected by the four selected alternative fumigants at both sites. In this study, microbial respiration showed a low sensitivity to detect changes in soil microbial activity due to repeated application of the standard MeBr + CP combination and alternative fumigants. This finding is in contrast with the high sensitivity of respiration measurements to treatment of soils with heavy metals and pesticides . Significant lower respiration rates in Oxnard soils fumigated with MeBr + CP compared to recently not fumigated control soils however, may indicate a decreased biological activity. Soil fumigation had no significant effect on microbial biomass C, and the results for microbial biomass N were inconsistent over the two experimental locations. Therefore, the effects of soil fumigation on total microbial biomass content provided little information on possible changes in the size of microbial populations. The overall low response of microbial biomass and respiration to repeated soil fumigation may be related to selected effect on sensitive microbial populations and the growth of resistant species. The latter may feed on cell debris, leading to restructuring of soil microbial populations as indicated elsewhere . Selected specialized bacteria may also use the fumigants as a source of carbon and energy, as documented for agricultural soils repeatedly subjected to MeBr fumigation . The effect of soil fumigation on the activities of dehydrogenase, β-glucosidase, acid phosphatase and arylsulfatase varied among the soil enzymes and within the two study sites. At the Watsonville site, soil fumigation with alternative fumigants generally had no significant effect on the activities of the four soil enzymes studied over the two year study period. Fumigation with MeBr + CP however severely affected the activities of β-glucosidase and acid phosphatase . These results suggest that biochemical reactions involved in organic matter degradation and P mineralization were affected by fumigation to a greater extent than were those reactions reflecting the general oxidative capabilities of microbial communities or involved in S mineralization in soils. In contrast, at the Oxnard site, β- glucosidase and acid phosphatase activities were relatively stable towards repeated soil fumigation, but dehydrogenase activity was significantly decreased by MeBr + CP. The reasons for these site-related variations in the response of soil enzyme activities to soil fumigants remain unclear. The two study sites showed very similar soil physical and chemical properties, such as clay and organic C contents. Variations may have occurred in the actual soil moisture content and temperature at the time of fumigation, which were proved to be crucial for the efficacy of pesticide applications . The results also suggest that the four alternative fumigants had no longer-term impact on enzyme reactions involved in organic matter turnover and nutrient cycling in soil. The inhibitory and/or activation effects of any compound in a soil matrix on enzyme activity are largely controlled by the reactivity of clay and humic colloids . The finding that MeBr + CP and the alternative fumigants led to a greater inhibition of the activities of the reference enzymes than that of soils suggests that free enzymes are more sensitive to soil fumigation than enzymes that are associated with the microbial biomass or enzymes adsorbed to clay or humic colloids. Ladd and Butler hypothesized that some enzymes are stabilized in the soil environment by complexes of organic and mineral colloids and therefore are partially protected from denaturation by fumigation. Similar results were observed for acid phosphatase, β-glucosidase and arylsulfatase in chloroform fumigated soils . Furthermore, reference enzymes were purified from one source for each protein, whereas soil enzymes derive from various sources leading to a set of isoenzymes [i.e., enzymes that catalyze the same reaction but may differ in origin, kinetic properties or amino acid sequencing ].