4 Enzymesand Microorganisms in theRhizosphere DavidC.Naseby Universityof Hertfordshire,Hatfield,Hertfordshire,England JamesM.Lynch UniversityofSurrey,Guildford,Surrey,England I.INTRODUCTION Althoughthetermrhizosphere wascoinedbyHiltner(1)todescribespecificallytheinter- actionbetweentherootsoflegumes andbacteria,theusageoftheterm hasbroadened. Todayweconsidertherhizosphere tobethezoneofinfluenceofall plantrootsystems, anditincludesthevariouscelllayersoftherootitself(the endorhizosphere),whichmicro- organismscancolonize;therootsurface(therhizoplane);andthe regionsurroundingthe root(theectorhizosphere).Therehas beensteadyprogressinourunderstanding ofthe rhizosphereduringthelast 100yearsandanincreasingrealizationthatitcanhavemany influencesoncrop productivity(Table1).However,therehasbeen a sudden increase in interest and investment in rhizosphere research in the past few years. This is largely due to the growth of biotechnology in relation to agriculture and, in particular, our increasing ability to manipulate plants by using recombinant deoxyribonucleic acid (DNA). Even with the genetic information of the plant modified, however, there will still be a highly influential microbial population associated with the roots. Therefore, the possibility exists that the balance between beneficial and harmful rhizosphere microorganisms might be improved not only by modification of the plant genotype but also by inoculation with useful and compatible microorganisms. The source of these useful bacteria and fungi could be either other soils and rhizospheres, hence amplifying the natural population, or introduc- tion of ‘‘foreign’’ organisms that have been genetically modified to elevate their useful properties and/or increase their competence to colonize the rhizosphere. Given current public opinion, the latter is unlikely in the near future. The general properties of rhizospheres have been described in several books (2–5). Not surprisingly two highly integrated microbe-plant root associations have attracted the most intense study. The first of these involves the activities of symbiotic dinitrogen-fixing bacteria. These bacteria express the nitrogenase enzyme complex and have received much attention in recent years as the various nif genes have been fully characterized (6–8). The other major group of microbe-plant symbionts in soil, the mycorrhiza, which promote the Copyright © 2002 Marcel Dekker, Inc. Table1InfluencesofRhizosphereMicroorganisms onCropProductivity BeneficialeffectsHarmfuleffects Nutrientandwaterup-Nutrientcompetition takeenhancement DinitrogenfixationDenitrification PhytohormoneproductionPhytotoxinproduction SymbiosisPathogenesis DiseaseandpestcontrolOxygendepletion SoilstabilizationDegradationofsoilstructure solubilizationanduptakeofphosphorusandotherplantnutrientsbyavarietyofbiotic andabioticmechanisms(9,10),havealsobeenstudiedextensively(seeChapter5). Rhizosphereenzymologyhasbeenparticularlyexploitedinbiomonitoringandim- pactanalysis.Mostattemptstomonitortheeffectsofmicroorganismsintherhizosphere havecenteredontheenumerationofspecificpopulations.However,forasignificantper- turbationtobemeasured,changesofbetween100%and300%arenecessarytoproduce asignificantimpact.Standardpopulationmeasurements,assessingthesurvivalanddis- seminationandeffectontotalindigenouspopulations,donotgiveanindicationofthe functioningoftheecosystem.Thereisverylittleliteratureregardingthefunctionalimpact ofmicrobesonecosystems,forinstance,theeffectuponnutrientcycling.Inthisreview functionalmethodologyisassessedforitsapplicationasanindicatoroftheimpactonthe soilecosystem.In1997arangeofsoilenzymeassayswasusedasanalternativeto,or incombinationwith,populationmeasurements(34).Theseappearquitesensitive,asim- pactsoflessthan20%onecosystemdisturbancecouldbesignificantlydetected.Enzyme functionsofthiskindintherhizospherearepotentiallyexploitableinprocessessuchas biocontrolandbioremediation,andthesearethemajorfocusofthisreview. II.SOILENZYMESASFUNCTIONALINDICATORS Soilenzymemeasurementsareextremelyimportantinassessingthestatusorthecondition ofthesoilenvironment.Thisisbecauseenzymesareessentialtothecyclingofnutrients insoilandarethuscriticaltotheavailabilityofnutrientstobothmicrobiotaandplants. Frequentlyenzymesareregulatedandexternalizedasaresponsetoexogenoussoilcondi- tions,suchasphosphatasesecretionduetophosphatedeficiency(37,38)orplantchitinase asaresultoffungalorinsectattack(11),andarethusgoodindicatorsofchangeor perturbation.Thesecondexampleshowsthatsoilenzymesarerespondingtothefunctional statusofthebiota,inadditiontothechemicalenvironmentasmeasuredbynutrientcycle measurements(Fig.1).Manyextracellularenzymesareadsorbedto,complexedwith,or entrapped within soil clays and humics (12). As a consequence, they may have a long- term stability and thus can provide an indication of the history of the sample and not just a snapshot from the time of sampling. However, as Figure 1 indicates, the ultimate assess- ment of the effect of the gene products is the plant bioassay (especially if the human perspective is considered), by which productivity and nutritional values are measured. Many enzymes are secreted actively into the soil environment by microbes and by Copyright © 2002 Marcel Dekker, Inc. Figure 1 A sequence illustrating the impact of a rhizosphere microorganism. The first functional product of the deoxyribonucleic acid (DNA) that can be monitored is messenger ribonucleic acid (RNA) which produces the gene products (enzymes and metabolites). plant roots and function by solubilizing nutrients; others are released into the soil by the lysis of microbial and plant cells. As mentioned, these extracellular enzymes often are immobilized by soil components and thus protected from degradation. There are several mechanisms by which enzymes can become immobilized in the soil, including adsorption on and within clay particles and organic matter, chemical complexing with organic matter, and entrapment within microbial polymers or soil aggregates (13). Once immobilized, the enzymes still may be active in the soil, but at variably reduced rates caused by factors such as steric hindrance, diffusional resistance, blocking of active sites, electrostatic effects, or reversible denaturation (14). If soil enzymes can be assessed and shown to vary with different soil treatments, then such measurements could be useful indicators of perturbation and of changes in functional diversity. Land management practices have been shown to have a significant influence on soil enzymes; for example, it has been demonstrated that cultivation can have a large effect on the soil arylsulfatase activity (15). Thus, long-term grassland soil cultivation (69 years) caused a 66% reduction in arylsulfatase activity, whereas cultivation of a forest soil for 47 years resulted in an 88% decrease. Large differences in sulfatase activity were found between a cultivated forest soil (63% decrease) and a similar forest soil that had been left fallow for 5 years (30% decrease). In the same study (15) changes in the kinetic properties of the soil sulfatases were investigated. After cultivation, a reduction was found in the V max (74% in the grassland Copyright © 2002 Marcel Dekker, Inc. soil and 90% in the forest soil) and the Michaelis constant (K m ), which decreased with the duration (years) of cultivation. Variations in K m values obtained from the forest and grassland soils suggest that the origins of the enzymes were different in the two soils. Therefore, such kinetic studies can be a good indicator of differences in soil enzymes with soil type and under very different soil management methods and may be sensitive enough to be applied to assessing the changes caused by less drastic soil treatments. Over- all, sulfatase activity was extremely sensitive to the soil management system and sulfatase may be one enzyme that is a good indicator of small-scale perturbations. A second example of the effect of land management practices on soil enzyme activi- ties is provided by the work of Jordan and associates (16). They evaluated microbial measurements (including acid and alkaline phosphatase activities) as indicators of soil quality in long-term cropping practices in historical fields. Differences in phosphatase activities among soils could be related to land management practices and soil properties (especially organic matter). For example, acid phosphatase activity was 150% higher in soil under continuous corn with no-till and receiving full fertility treatment than under conventional tillage with no fertility treatment. Alkaline phosphatase activity was 50% higher in soil under continuous corn with full fertility treatment than without the fertility treatment. This study again shows that soil enzyme activities can change greatly after the large perturbations caused by soil management practices and thus have great potential as a general indicator of smaller-scale perturbation. In another study concentrating on the impact of crop and land management systems on soil enzyme activities from a coconut-based multistoried cropping system (17), de- creases in urease and an unspecified phosphatase (but not dehydrogenase) with increasing depth in rhizosphere soil were found. Differences were found also in urease and phospha- tase with the different crops grown. Cocoa and pineapple produced contrasting results to the coconut rhizosphere (higher phosphatase activity in the coconut rhizosphere and higher urease activity with multistoried systems over a coconut monocrop). Depth and crop sys- tem appear to have a large influence on rhizosphere enzyme activities. The effect of pesticides on the activity of various soil enzymes has been investigated by a number of researchers (18,19). A prime example of this is the work of Satpathy and Behera (20), who examined the effect of malathion on cellulase, protease, urease, and phosphatase activities in a tropical grassland soil. They found that the significantly de- pressed cellulase and protease activity recovered to almost the same level as that of the control 21 days after malathion application, whereas urease and, to a larger extent, phos- phatase activities did not show such a rapid recovery. A similar situation to the addition of pesticides to soil is the input of inorganic pollutants, which can have major effects not just on enzymes but on the whole soil ecosys- tem. Decreases in cellulase activity were found with increasing sulfur, nitrogen, and heavy metal pollution (21). The reduction in cellulase activity was correlated with a decrease in respiration along the same pollution gradient. However, cellulase activity was a more precise indicator of the level of pollution than were respiration measurements. III. MICROBIAL INOCULATION AND SOIL ENZYME ACTIVITIES A proposed target for genetic manipulation is the insertion or enhancement of genes encod- ing specific enzymes; a prime candidate for this is chitinase because of its postulated role in the biocontrol of fungal crop pathogens. Ridout and colleagues (22) investigated the Copyright © 2002 Marcel Dekker, Inc. protein production induced in a Trichoderma species when cell wall fragments of the crop plant pathogen Rhizoctonia solani (to which the Trichoderma species was an antagonist) were used as the sole carbon source. Both β-glucanase and chitinase were found to be important components of the inducible extracellular proteins analyzed by electrophoretic profiles. The sequence of chitin degradation (and pathogen biocontrol) was extremely complex with several other inducible enzymes probably involved. Much of the interest in the enzymology of Trichoderma sp. pathogen biocontrol has focused on chitinase and 1,3-β-d-glucanase, although proteases could be involved (22,23). Furthermore, many authors have suggested that chitinase may be the most important en- zyme in biocontrol by Trichoderma sp., and because the enzyme can be purified and characterized readily (24), it has been a good target for transformation (25). As yet, how- ever, it has not been possible to demonstrate that introduction of the gene into a nonproduc- ing strain increases biocontrol potential. However, much has been learned about gene expression and its regulation during mycoparasitism of fungal pathogens by Trichoderma sp. by using gene probes and markers (26–28). Other rhizosphere microorganisms produce chitinase and exert biocontrol effects; notable among these are the actinomycetes (29). To detect these chitinase genes, a chiA DNA probe is available (30). It should be remembered, however, that detection of a gene does not necessarily mean the gene will be expressed, and therefore, in the example here, a positive signal from the probe does not necessarily mean that chitinase is expressed— merely that the genotype or potential exists. The situation in which genes encoding enzymes (especially extracellular) are the target of manipulation gives an added emphasis to the use of soil enzyme activities as a measure of perturbations caused by the introduction of such microorganisms into the soil and rhizosphere. In an attempt to understand the effect of manipulated enzyme production on a soil microorganism, one study involved rhizosphere growth of Pseudomonas solana- cearum, which was genetically altered in extracellular enzyme production (endopolygalac- touronase A and endoglucanase) (31). The strains that had been enhanced in terms of enzyme production had greatly reduced fitness in the rhizosphere. However, the authors did not use the opportunity to study the effect on soil extracellular enzyme activity with the inoculation of strains with such functional modifications. There are few data regarding the use of soil enzymes as a measure of perturbations caused by the introduction of extraneous microorganisms into the soil or the rhizosphere, and much of the available information is contradictory. Doyle and Stotzky (32) evaluated a number of enzymes, including arylsulfatase, dehydrogenase, and acid and alkaline phos- phatase, for the detection of changes in the microbial ecological characteristics of the soil caused by the introduction of Escherichia coli but found no consistent significant differ- ences in any of the enzyme activities. It must be noted, however, that this experiment (as with a great many others) did not reflect ‘‘ecologically relevant’’ conditions, as the strains used were not natural soil organisms and the experiment was conducted in bulk soil in the absence of plants. Commercial inoculants often are designed to live and perform functions in the rhizo- sphere and respond to root exudates (a major source of carbon and nutrients). Therefore, it is not surprising that the work of Doyle and Stotzky (32) found few effects upon enzyme activities. The survival and metabolic activity of the E. coli strain were likely to be low since there was no additional substrate and indigenous carbon would have been unsuitable or utilized by the preexisting microflora. Furthermore, E. coli is not a soil organism and is unlikely to establish a viable population after introduction into soil. In contrast, Copyright © 2002 Marcel Dekker, Inc. Mawdsley and Burns (33) introduced a soil Flavobacterium species into the rhizosphere of wheat. They found that the microbial inoculant caused increased α-galactosidase, β- galactosidase, α-glucosidase, and β-glucosidase activities in the more ecologically and agriculturally relevant conditions of the wheat rhizosphere. A number of simple enzyme assays for the detection of perturbations resulting from different soil treatments, including the introduction of a modified Pseudomonas fluo- rescens SBW25 strain and substrate amendments, have been designed (34). The aim of the experiment was to deduce whether these assays were sensitive enough to measure perturbations caused by microbial inoculation. Specific attention was paid to the validation of soil biochemical techniques as a method of monitoring the effects of inoculation. Differ- ences in wheat rhizosphere soil biomass (measured by adenosine triphosphate [ATP] con- tent) and several key soil enzyme activities with microbial inoculation and/or modified conditions (addition of the enzyme substrates, chitin, urea, and glycerophosphate, to soil) were measured. Microbial biomass, as well as enzyme activities (with the exception of that of acid phosphate), decreased with depth. The addition of a substrate mixture of urea, colloidal chitin, and glycerophosphate to soil significantly increased N-acetyl glucosaminidase, chi- tobiosidase, arylsulfatase, and urease activities but did not cause a change in acid and alkaline phosphatase and phosphodiesterase activities. Inoculation of wheat seeds with P. fluorescens resulted in significant increases in rhizosphere chitobiosidase and urease activi- ties at 5- to 20-cm depth and a significant decrease in alkaline phosphatase activity. The 0- to 5-cm depth activities were unchanged. Inoculation with P. fluorescens in combination with the substrate mixture had effects opposite to those of treatments without substrate mix addition: chitobiosidase, arylsulfatase, and urease activities were significantly lower and alkaline phosphatase was significantly higher at the 5- to 20-cm depth interval with inoculation of bacteria. Biomass values for the combined bacteria and substrate mix treat- ment were significantly higher than for the substrate mix alone treatment. IV. SOIL ENZYME ACTIVITIES AND GENETICALLY MODIFIED ORGANISMS Naseby and Lynch (35) used enzymatic analysis, combined with microbial population measurements and other soil indicators, to investigate the effect of wild-type and geneti- cally modified microbes in the rhizosphere. They studied whether the impact of a P. fluo- rescens strain, a genetically modified microbe (GMM) altered for kanamycin resistance and lactose utilization, could be enhanced by adding lactose and kanamycin to the soil (prior to/subsequent to amendment). Lactose addition decreased the shoot-to-root ratio and both soil amendments increased the populations of total culturable bacteria and the inoculated GMM. Only kanamycin perturbed the bacterial community dynamics, causing a shift toward slower-growing organisms. The community structure with the GMM inocu- lum, in the presence of kanamycin, showed the only impact of the GMM compared to that the wild-type inoculum. The shift toward K strategists (i.e., slower-growing organ- isms) found in the other kanamycin-amended treatments was reversed with the GMM inoculation. Lactose amendment increased acid and alkaline phosphatase, phosphodiester- ase, and carbon cycle enzyme activities, whereas the kanamycin addition affected only the alkaline phosphatase and phosphodiesterase activities. None of the soil enzyme activi- ties was affected by the GMM under any of the soil amendments. Copyright © 2002 Marcel Dekker, Inc. Althoughthisstudyinvolvedtheuseofageneticallymodifiedmicrobe,themodifi- cationswerenotintendedtohaveafunctionalimpact;theywereinsertedasgeneticmark- ers.Asecondstudycomparingtheeffectofthesamegeneticallymarkedstraintothatof afunctionallymodifiedstrainshowedeffectsthataremoreinteresting(36).Theaimof thisworkwastodeterminetheimpactintherhizosphereofwildtypealongwithfunction- allyandnonfunctionallymodifiedPseudomonasfluorescensstrains.Thewild-typeF113 straincarriedageneencodingtheproductionoftheantibiotic2,4-diacetylphloroglucinol (DAPG),usefulinplantdiseasecontrol,andwasmarkedwithalacZYgenecassette.The firstmodifiedstrainwasafunctionalmodificationofstrainF113withrepressedproduction ofDAPG,creatingtheDAPGnegativestrainF113G22.Thesecondpairedcomparison wasanonfunctionalmodificationofwild-type(unmarked)strainSBW25,constructedto carrymarkergenesonly,creatingstrainSBW25EeZY-6KX. Significantperturbationswererecordedintheindigenousbacterialpopulationstruc- ture;theF113(DAPGϩ)straincausedashifttowardslower-growingcolonies(Kstrate- gists)comparedwiththenon-antibiotic-producingderivative(F113G22)andSBW25 strains.TheDAPGϩstrainalsosignificantlyreduced,incomparisonwiththoseofthe otherinocula,thetotalPseudomonassp.populations,butdidnotaffectthetotalmicrobial populations.ThesurvivalofF113andF113G22wasanorderofmagnitudelowerthan thatoftheSBW25strains.TheDAPGϩstraincausedasignificantdecreaseintheshoot- to-rootratioincomparisontothatofthecontrolandotherinoculants,indicatingplant stress.F113increasedsoilalkalinephosphatase,phosphodiesterase,andarylsulfataseac- tivities(Table2)comparedtothoseofthecontrols.Theotherinoculareducedthesame enzyme activities when compared to the control. In contrast, the β-glucosidase, β-galactos- idase, and N-acetyl glucosaminidase activities decreased with the inoculation of the DAPGϩstrain(Table1).Theseresultsindicatethatsoilenzymesaresensitivetothe impact of GMM inoculation. Increased available (soluble) inorganic phosphate is known to decrease soil phospha- tase activity (37,38). Therefore, the F113 (DAPGϩ) strain must have caused a decrease in the available phosphate, thus causing an overall increase in activity. The decrease in available P may have taken the form of an increase in the available carbon in the rhizo- sphere (by stimulation of root exudation or leakage, as there was a decrease in the shoot/ root ratio). Other studies have highlighted changes in root exudation caused by biocontrol P. fluorescens strains (39). Therefore, increasing the ratio of C to P available would in- crease the microbial P demand. Inverse trends were found with the C and N cycle enzymes in comparison to the general trend found in the P and S cycle enzymes. The F113 (DAPGϩ) strain was associ- ated with the lowest acid β-galactosidase activity, which was significantly lower than that of the SBW25 WT treatment. The F113 (DAPGϩ) strain produced the lowest β-glucosi- dase activity, which was significantly smaller than that of the control, the SBW25 WT, and the F113 G22 treatments, which all had similar activities. As with the β-galactosidase and β-glucosidase activities the F113 (DAPGϩ) strain produced the lowest N-acetyl glu- cosaminidase activity. This was a significantly lower activity than that of the control, the SBW25 WT, and the F113 G22 treatments, which all had comparable activities. All three carbon cycle enzyme activities, therefore, also indicate an increase in carbon availability. Subsequent work with a genetically modified DAPG overproducer found effects similar to those of the wild-type DAPG producer (40). Further, the carbon fractions in the rhizosphere of pea plants inoculated with strains F113 and F113 G22 were examined (41). Both strains significantly increased the water- Copyright © 2002 Marcel Dekker, Inc. Table 2 Soil Enzyme Activities in the Rhizosphere of Pea Plants Inoculated with Wild-Type and Genetically Modified Pseudomonas fluorescens Strains Acid Alkaline Acid Treatment a phosphatase b phosphatase b Phosphodiesterase b Arylsulfatase b β-galactosidase b β-glucosidase b NAGase b Control 7.91ab Ϯ 0.40 1.23bc Ϯ 0.15 0.11a Ϯ 0.018 0.09a Ϯ 0.01 0.52ab Ϯ 0.04 1.02b Ϯ 0.10 0.32b Ϯ 0.03 F113 G22 8.24ab Ϯ 0.69 0.70a Ϯ 0.16 0.09a Ϯ 0.03 0.08a Ϯ 0.02 0.1ab Ϯ 0.05 1.02b Ϯ 0.14 0.29b Ϯ 0.03 F113 7.06a Ϯ 0.40 1.52c Ϯ 0.13 0.17b Ϯ 0.01 0.16b Ϯ 0.02 0.45a Ϯ 0.04 0.62a Ϯ 0.07 0.18a Ϯ 0.02 a Treatments: control, no inocula; F 1113 G22, inoculated with lacZY marked DAPGϪ (Tn5 mutated) P. fluorescens F113 G22; F113, inoculated with lacZY marked DAPGϩ P. fluorescens F113. b mg pNP Released g Ϫ1 dry soil. Standard errors for means (n ϭ 7) indicated. Values followed by the same letter within a column are not significantly different at p ϭ 0.05 level. Copyright © 2002 Marcel Dekker, Inc. solublecarbohydratesandthetotalwater-solublecarbonintherhizospheresoil(Table 3).StrainF113significantlyincreasedthesoilproteincontentrelativetothatofthecontrol butnotinrelationtotheF113G22treatment.TheF113treatmenthadasignificantly greaterorganicacidcontentthanthecontrolandF113G22treatments;theF113G22 treatmentalsohadsignificantlygreatercontentthanthecontrol.Bothinocularesulted insignificantlylowerphosphatecontentthanthecontrol.Bothinoculaincreasedcarbon availability;however,antibioticproductionbystrainF113reducedtheutilizationofor- ganicacidsreleasedfromtheplant,resultingindifferingeffectsofthetwostrainson nutrientavailability,plantgrowth,soilenzymeactivities,andPseudomonassp.popula- tions. Thesestudieshighlighttheimportanceoftheuseofsoilenzymeactivitiesinimpact studies.Theyalsoillustratehowthecombinationofanumberofenzymaticmeasurements fromdifferentnutrientcyclescanbeusedasadiagnostictooloftheprocessesinvolved insuchperturbations.Theenzymaticmethodsworkedwellinsmallmicrocosmsandlarge intactsoilcoreswhereenvironmentalvariationisminimized;thesamemethodsthenwere testedinfieldscaletrialsoftheF113strain. SoilenzymeactivitieswereusedtoinvestigatetheimpactofstrainF113inthe rhizosphereoffield-grownsugarbeet(42).Thereweredistincttrendsinrhizosphereen- zymeactivitiesinrelationtosoilchemicalcharacteristics(measuredbyelectroultrafiltra- tion).Forexample,theactivitiesofenzymesfromthephosphoruscycle(acidphosphatase, alkalinephosphatase,andphosphodiesterase)andofarylsulfatasewerenegativelycorre- latedwiththeamountofreadilyavailableP(Table4),whereasureaseactivitywasposi- tively correlated with available P. Significant correlations between electroultrafiltration nutrient levels and enzyme activity in the rhizosphere were obtained, highlighting the usefulness of enzyme assays to document variations in soil nutrient cycling. Contrary to the results of previous microcosm studies, which did not investigate plants grown to matu- rity, the biocontrol inoculant had no obvious effect on enzyme activity or on soil chemical characteristics in the rhizosphere. The results show the importance of homogenous soil microcosm systems, used in previous work, in risk assessment studies in which inherent soil variability is minimized and an effect of the pseudomonad on soil enzymological features could be detected. This study also highlights the fact that the effect of spatial variability is far greater than the effect of any microbial inocula. Following this study of the effects of a biocontrol agent on soil enzyme activities in a field trial, the soil enzyme methodology was used to assess the impact of genetically modified plants on soil biochemical features (D. C. Naseby, A. Greenland, and J. M. Table 3 Carbon Fractions of Pea Rhizosphere Soil Inoculated with DAPG-Producing and -Nonproducing Pseudomonas fluorescens Strain F113 C fraction a Control b F113 G22 b F113 b Carbohydrates (H 2 O) 139.44a Ϯ 8.57 200.04b Ϯ 8.36 209.41b Ϯ 8.29 Water-soluble C 1855.32a Ϯ 139.7 2731.78b Ϯ 77.73 2669.73b Ϯ 54.92 Protein 56.16a Ϯ 4.78 69.56ab Ϯ 4.66 78.38b Ϯ 5.88 Organic acids 6.09a Ϯ 0.36 7.18b Ϯ 0.38 9.92c Ϯ 0.66 a Expressed as ppm g Ϫ1 dry soil. Standard errors for means (n ϭ 8) indicated. Values followed by the same letter within a column are not significantly different at p ϭ 0.05 level. b Treatments: control, no inocula; F113 G22, inoculated with lacZY marked DAPGϪ (Tn5 mutated) P. fluo- rescens F113 G22; F113, inoculated with lacZY marked DAPGϩ P. fluorescens F113. Copyright © 2002 Marcel Dekker, Inc. Table 4 Coefficients of Correlation Between Alkaline Phosphatase Activity and EUF Levels (Soil Chemicals) in a Sugar Beet Field Trial Inoculated with Pseudomonas fluorescens Strain F113 Activity Mg Treatment (mg pNP g Ϫ1 soil) Org N Ca P K (µgg Ϫ1 soil) Na Mn pH NO 3 Non Rhiz 1.658 17 519 38 118 19 32 0.5 7.0 4.0 Cont 0.671 17 549 53 388 27 59 1.2 7.2 7.2 Cont 2.261 12 480 30 208 19 27 1.2 6.9 4.7 Cont 1.624 17 396 41 210 19 24 0.6 6.9 5.4 F113 1.241 15 665 44 211 27 15 1.6 6.9 6.1 F113 2.526 14 593 31 197 25 23 1.5 7.0 5.8 F113 1.878 21 548 31 321 25 45 1.4 7.0 9.0 r value Ϫ0.348 Ϫ0.14 Ϫ0.95** Ϫ0.51 Ϫ0.40 Ϫ0.49 Ϫ0.11 Ϫ0.53 Ϫ0.24 ** Significant at P ϭ 0.01; n ϭ 7. Copyright © 2002 Marcel Dekker, Inc. [...]... plant chitinase induced by fungi Prog Biochem Biophys 27: 40 44 , 2000 12 RG Burns Enzyme-activity in soil—location and a possible role in microbial ecology Soil Biol Biochem 14: 423 42 7, 1982 13 HH Weetall Immobilised enzymes and their application in the food and beverage industry Process Biochem 10:3– 24, 1975 14 G Haska Activity of bacteriolytic enzymes adsorbed to clays Microb Ecol 7:331– 341 , 1981... measured and with the available soil nutrients However, there was little difference between the enzyme activities in the rhizosphere of the GM and non-GM plants The major factor in uencing the enzyme activities and soil nutrients between the two sampling days was the soil moisture content, which was increased by overnight rain Therefore, in this field trial, the differences between soil enzyme activities were... laccase-type polyphenol oxidase in the rhizosphere of rice, a factor that may be involved in the colonization of roots (49 ) However, endochitinase continues to be implicated in the biocontrol of Pythium ultimum by Trichoderma spp (50,51) Immunological detection of diazotrophic enterobacterial strains with growth-promoting properties, using the double-antibody sandwich enzyme-linked immunosorbent assay,... fluorescens on the root exudates of two tomato mutants differently sensitive to Fe chlorosis Plant Soil 144 :167–176, 1992 40 DC Naseby, JM Lynch Effect of 2 ,4 diacetylphloroglucinol producing, overproducing and non-producing Pseudomonas fluorescens F113 in the rhizosphere of pea Microbial Ecology 2001 41 DC Naseby, J Pascual, JM Lynch Carbon fractions in the rhizosphere of pea inoculated with 2 ,4 diacetylphloroglucinol... Grundunging und Brache Arb Dtsch Landwist Ges 98:59–78, 19 04 2 EA Curl, B Truelove The Rhizosphere Berlin: Springer-Verlag, 1986 3 JM Lynch, ed The Rhizosphere Chichester: John Wiley, 1990 4 R Pinton, Z Varanini, P Nannipieri, eds The Rhizosphere: Biochemistry of Organic Substances at the Soil-Plant Interface New York: Marcel Dekker, 2000 5 PB Tinker, PH Nye Solute Movement in the Rhizosphere New York: Oxford... null line (not expressing antifungal proteins) Lynch, 1999 unpublished data) The two modified oil seed rape lines used (Brassica napus var Westar) produced small cysteine-rich proteins with antifungal activity, specifically expressing either the DmAMP1 gene from Dahlia merckii or the AceAMP1 gene from Allium cepa The field trial consisted of these transgenic lines compared to a number of controls, their... had the attention that they warrant Enzyme production in field soil is attracting interest In a forested landscape (53), measurements of β-glucosidase, chitinase, phenol oxidase, and acid phosphatase with explanatory path analysis indicated that the four enzymes could help resolve spatial dependencies at a range of scales and could also be used to develop a scale-independent metric to be used for the. .. supplies the carbon and energy for the microorganisms to produce the bioremediation, although plants themselves may also have cyanide-catabolizing potential The harnessing and exploitation of rhizosphere enzymes are at a very early stage of development; however, such use of rhizosphere enzymes seems set to advance rapidly Much of the emphasis of the most recent papers on rhizosphere enzymes has concerned the. .. produce enzymes that have the capacity to catabolize a wide range of organic pollutants Microbial dehalogenation is described in detail in Chapters 18 and 19, but of special interest are hydrogen cyanide and other nitriles Not only is the xenobiotic source of cyanides from industrial pollution important, but some microorganisms themselves produce cyanides in the rhizosphere (45 ), and these are potentially... diacetylphloroglucinol producing and non-producing Pseudomonas fluorescens F113 J Appl Microbiol 87:173–181, 1999 ¨ 42 DC Naseby, Y Moenne-Loccoz, J Powell, F O’Gara, JM Lynch Soil enzyme activities in the rhizosphere of field-grown sugar beet inoculated with the biocontrol agent Pseudomonas fluorescens F113 Biol Fertil Soils 27:39 43 , 1998 43 JM Lynch, A Wiseman, eds Environmental Biomonitoring: The Biotechnology . Inc. Althoughthisstudyinvolvedtheuseofageneticallymodifiedmicrobe,themodi - cationswerenotintendedtohaveafunctionalimpact;theywereinsertedasgeneticmark- ers.Asecondstudycomparingtheeffectofthesamegeneticallymarkedstraintothatof afunctionallymodifiedstrainshowedeffectsthataremoreinteresting(36).Theaimof thisworkwastodeterminetheimpactintherhizosphereofwildtypealongwithfunction- allyandnonfunctionallymodifiedPseudomonasfluorescensstrains.Thewild-typeF113 straincarriedageneencodingtheproductionoftheantibiotic2 , 4- diacetylphloroglucinol (DAPG),usefulinplantdiseasecontrol,andwasmarkedwithalacZYgenecassette .The firstmodifiedstrainwasafunctionalmodificationofstrainF113withrepressedproduction ofDAPG,creatingtheDAPGnegativestrainF113G22.Thesecondpairedcomparison wasanonfunctionalmodificationofwild-type(unmarked)strainSBW25,constructedto carrymarkergenesonly,creatingstrainSBW25EeZY-6KX. Significantperturbationswererecordedintheindigenousbacterialpopulationstruc- ture;theF113(DAPGϩ)straincausedashifttowardslower-growingcolonies(Kstrate- gists)comparedwiththenon-antibiotic-producingderivative(F113G22)andSBW25 strains.TheDAPGϩstrainalsosignificantlyreduced,incomparisonwiththoseofthe otherinocula,thetotalPseudomonassp.populations,butdidnotaffectthetotalmicrobial populations.ThesurvivalofF113andF113G22wasanorderofmagnitudelowerthan thatoftheSBW25strains.TheDAPGϩstraincausedasignificantdecreaseintheshoot- to-rootratioincomparisontothatofthecontrolandotherinoculants,indicatingplant stress.F113increasedsoilalkalinephosphatase,phosphodiesterase,andarylsulfataseac- tivities(Table2)comparedtothoseofthecontrols.Theotherinoculareducedthesame enzyme. would in- crease the microbial P demand. Inverse trends were found with the C and N cycle enzymes in comparison to the general trend found in the P and S cycle enzymes. The F113 (DAPGϩ) strain was. Inc. Althoughthisstudyinvolvedtheuseofageneticallymodifiedmicrobe,themodi - cationswerenotintendedtohaveafunctionalimpact;theywereinsertedasgeneticmark- ers.Asecondstudycomparingtheeffectofthesamegeneticallymarkedstraintothatof afunctionallymodifiedstrainshowedeffectsthataremoreinteresting(36).Theaimof thisworkwastodeterminetheimpactintherhizosphereofwildtypealongwithfunction- allyandnonfunctionallymodifiedPseudomonasfluorescensstrains.Thewild-typeF113 straincarriedageneencodingtheproductionoftheantibiotic2 , 4- diacetylphloroglucinol (DAPG),usefulinplantdiseasecontrol,andwasmarkedwithalacZYgenecassette .The firstmodifiedstrainwasafunctionalmodificationofstrainF113withrepressedproduction ofDAPG,creatingtheDAPGnegativestrainF113G22.Thesecondpairedcomparison wasanonfunctionalmodificationofwild-type(unmarked)strainSBW25,constructedto carrymarkergenesonly,creatingstrainSBW25EeZY-6KX. Significantperturbationswererecordedintheindigenousbacterialpopulationstruc- ture;theF113(DAPGϩ)straincausedashifttowardslower-growingcolonies(Kstrate- gists)comparedwiththenon-antibiotic-producingderivative(F113G22)andSBW25 strains.TheDAPGϩstrainalsosignificantlyreduced,incomparisonwiththoseofthe otherinocula,thetotalPseudomonassp.populations,butdidnotaffectthetotalmicrobial populations.ThesurvivalofF113andF113G22wasanorderofmagnitudelowerthan thatoftheSBW25strains.TheDAPGϩstraincausedasignificantdecreaseintheshoot- to-rootratioincomparisontothatofthecontrolandotherinoculants,indicatingplant stress.F113increasedsoilalkalinephosphatase,phosphodiesterase,andarylsulfataseac- tivities(Table2)comparedtothoseofthecontrols.Theotherinoculareducedthesame enzyme