CSIRO PUBLISHING www.publish.csiro.au/journals/ijwf International Journal of Wildland Fire, 2005, 14, 355–363 Functional diversity of the microbial community in Mediterranean maquis soils as affected by fires Rosaria D’AscoliA,C , Flora A. RutiglianoA , Raffaele A. De PascaleA , Anna GentileB and Amalia Virzo De SantoB A Dipartimento di Scienze Ambientali, Seconda Università degli Studi Napoli, Via Vivaldi 43, 81100 Caserta, Italy. B Dipartimento di Biologia Vegetale, Università degli Studi di Napoli Federico II, Via Foria 223, 80139 Napoli, Italy. C Corresponding author. Telephone: +39 823 274644; fax: +39 823 274605; email: rosaria.dascoli@unina2.it Abstract. Fire is a disturbance in the Mediterranean region associated with frequent drought periods, and can affect the soil microbial community, which plays a fundamental role in nutrient cycling. In the present study the effect of low- and high-severity experimental fires on the soil microbial community was evaluated in an Italian Mediterranean maquis. Burned and unburned soils were compared for functional diversity, specific activities, microbial biomass, fungal mycelia and fungal fraction of microbial carbon, during the first year after fire. In the first week after fire, changes in the functional diversity were observed in burned soils, differing also between low- and high-severity fires. Respiration responses to specific organic compounds were generally lower in burned soils during the whole study period, with a percentage of changed responses from 2 to 70%. The general reduction in burned soils of the fungal fraction of microbial carbon (19–61%) and active mycelia (16–55%), together with the increase in microbial biomass carbon (29–42%) during the first 3 months after fire, suggest a larger and longer effect of fire on fungi than on bacteria. The results indicate a rapid recovery of functional diversity in soil after burning despite the persistent reduction of microbial community activity and the change in its structure. Additional keywords: catabolic evenness; fungal mycelia; microbial biomass. Introduction Fire represents a disturbance factor that plays an important ecological role in the evolution, dynamics and distribution of vegetation in the world (Gill et al. 1981; Wright and Bailey 1982; Booyesen and Tainton 1984) as it influences structure (Schaefer 1993; Laterra 1997; Whittle et al. 1997) and species composition (Trabaud and Lepart 1980; Agrawal 1990; Boo et al. 1997) of the plant community. Since the Neolithic, large natural habitats were burned to convert them to cultivated or grazed fields (Le Coz 1990), but in some habitats fire is also a natural ecological factor. In the Mediterranean region, regular drought periods favour the combustion processes, so fires are frequent (Trabaud and Grandjanny 2002). Moreover, because of the climatic changes in progress the risk of fires is destined to increase in the various fire-prone regions (Beer et al. 1988; Flannigan and Van Wagner 1991; Torn and Fried 1992). Fire is a key factor in maintaining the structure and functioning of the Mediterranean ecosystems and has © IAWF 2005 played a fundamental role in the evolution of resistance characters in Mediterranean plants (Naveh 1975). Many authors have studied fire effects on Mediterranean flora and wildlife, with particular focus on the diversity of species (Trabaud and Lepart 1980; Prodon et al. 1987; Hobbs and Atkins 1990). Likewise, fire effects on soil microorganisms, involved in ecosystem processes such as decomposition and nutrient cycling, may also be highlighted by evaluating species and functional diversity of the microbial community, which is fundamental for the functional capability of soils (Giller et al. 1997). It has been hypothesized that declines in the species and functional diversity of soil organisms may negatively affect the resistance of soils to disturbance (Brussaard et al. 1997; Giller et al. 1997). As reported by Bengtsson (1998) ‘the main importance of diversity is not that it in itself has a function in ecosystems, but that high diversity implies that there is a source of new species performing functions or 10.1071/WF05032 1049-8001/05/040355 356 R. D’Ascoli et al. Int. J. Wildland Fire ecosystem services as human needs or environmental conditions change’. Nevertheless, until now it has not been possible to assess completely the species diversity of the soil microbial community using current culture-based, molecular and biochemical methods (Zak et al. 1994; Trevors 1998). Moreover, community structure does not provide information about the functional diversity of the microbial community, which is a component of overall diversity in soil representing an important indicator of soil functioning (Degens 1999). In fact, the microorganisms are not necessarily functionally active in soils (van Veen and Heijnen 1994; Trevors 1998). Therefore, functional diversity may provide an ecologically relevant measure of microbial diversity (Zak et al. 1994). All of the microbial activities involved in soil functions, such as decomposition, nutrient transformations and plant growth promotion–suppression, are part of soil functional diversity (Giller et al. 1997), with the catabolic diversity of heterotrophic microorganisms (i.e. diversity of decomposition functions) representing an important component of soil functional diversity. Until now, studies carried out on Mediterranean soils affected by fire have not taken into account functional diversity of the soil microbial community, but only its growth and activity (Fritze et al. 1993; Pietikäinen and Fritze 1993; Rutigliano et al. 1995, 2002a; Dumontet et al. 1996; Rahkonen et al. 1999; Pietikäinen et al. 2005). Nevertheless, the catabolic diversity of the microbial community, together with other biological parameters such as microbial activity and biomass, can provide more complete information on the status of the microbial community after burning. In fact, it was shown that low values of catabolic diversity resulting from intensive land uses can affect the resistance of soils to stress or disturbance, i.e. the ability of microbial catabolic diversity to remain constant when permanent or limited-in-time events occur (Degens et al. 2001). It is therefore important to understand whether fire can affect the soil resistance. The aim of this study was to evaluate, in a Mediterranean maquis area, the effect of fire on functional diversity of the soil microbial community as well as on microbial activity, total microbial biomass and active fungal mycelia, which are the parameters usually used to describe the microbial community. Therefore, experimental fires with different severity have been carried out in CastelVolturno Nature Reserve and their effects on microbial community were assayed during the first year after fires. Functional diversity of the soil was determined both as catabolic response profile, that is short-term respiratory response of soil due to addition of several simple organic compounds, and as catabolic evenness (Degens et al. 2000). In fact, as it is generally accepted that diversity reflects richness and evenness, and given that it is impracticable to measure the immense richness of microbial functions in soil, we have measured only some catabolic functions in order to calculate catabolic evenness from catabolic response profile using the Simpson–Yule index (Magurran 1988). On the other hand, each respiratory response of soil to the addition of one organic compound can be also considered one specific microbial activity. It has to be emphasized that the growth of the fungal component in the soil microbial community can represent an indicator of stress or disturbance. Fungi are sensitive to the shortage of organic matter and nutrients in the soil (Nordgren et al. 1983; Iovieno et al. 1996), water availability, pH increase (Nordgren et al. 1983), temperature increase (Rundel 1981; Pietikäinen et al. 2005) and changes due to fire (Pietikäinen and Fritze 1995). Thus, in order to establish the relative importance of fungi within the total microbial community, we have also calculated the fungal fraction of microbial carbon. Materials and methods Study area and experimental design The study was carried out in the Castel Volturno Nature Reserve, a flat coastal area in south-western Italy subjected to a typically Mediterranean climate, with rainy autumn and winter and long summer drought periods that create a high fire risk during the summer. Since 1974, the Forest Service management has prevented vegetation cutting or grazing, so that fire has become the main factor affecting vegetation dynamics (Esposito et al. 1998). In the reserve, the vegetation is characterized by patches of low and high maquis with scattered Pinus trees planted by foresters. In this area frequent shrub species of the maquis are Quercus ilex L., Phillyrea sp. pl., Myrtus communis L., Arbutus unedo L., Pistacia lentiscus L., Rhamnus alaternus L. and Cistus sp. pl. (Esposito et al. 1999). Small gaps in the shrub cover of the maquis are dominated by herbs and bryophytes. A few stands of Q. ilex and plantations of Pinus pinea L. were also present (Esposito et al. 1999). The soil at the experimental site is a Calcaric Arenosol, according to the FAO classification (FAO 1998; di Gennaro 2002). In the Castel Volturno Nature Reserve, an area covering ∼1300 m2 dominated by Cistus salvifolius L., Rosmarinus officinalis L., M. communis and Phillyrea angustifolia L. was selected and divided by firebreaks deprived of vegetation into nine plots each of 50 m2 . On 3 July 2000 experimental fires with different severity were carried out. Three plots were burned with low-severity fire, three plots were burned with high-severity fire and three plots remained unburned to serve as a control. In order to carry out fires with lowand high-severity, different fuel loads were used. For the low fire-severity plots, the vegetation cover was reduced by cutting to obtain an average value of 2 kg m−2 of almost evenly distributed biomass. For the high fire-severity plots, wood material taken from the surrounding vegetation was added to the canopy cover to obtain an average value of 4 kg m−2 of almost evenly distributed biomass. These fuel Microbial functional diversity in burned soils load values were identical to the values used by Molina and Llinares (1998) to achieve contrasting levels of fire intensity. In fact, they showed that an increase of fuel load from 2 to 4 kg m−2 in maquis vegetation resulted in roughly a doubling of the net radiant heat per unit area (Molina and Llinares 2001). The biomass values utilized in the present study to obtain the different-severity fires were comparable with real biomass values found in low and high maquis of the Castel Volturno Nature Reserve. Others authors (De Luis et al. 2004) found biomass values similar to values reported in this study in mature communities of Mediterranean gorse shrublands of Spain (i.e. 3–4 kg m−2 ). Moreover, Olson (1981) reports that, in Mediterranean-type shrublands, fuel combustion during fire events varies from 1 to 5 kg m−2 . Thus, the experimental fires were comparable, with respect to quality and quantity of fuel, with fires occurring in this or other similar shrub communities of the Mediterranean region. During the fire, the flames seemed almost evenly distributed on the plots. However, it has to be emphasized that, although the fuel load was almost homogeneously distributed, in the Mediterranean shrublands not all the ground is covered by plants. The spaces between the plants do not have the same amount of biomass, nor is the height of the fuel the same (Molina and Llinares 2001). Moreover, even in homogeneous stands of Mediterranean-type shrublands, fire intensity can vary depending on plant distribution and on the vagaries of the fire producing a distinct fire pattern (Moreno and Oechel 1994). After burning, the plots affected by the low-severity fire showed remnants of unburned vegetation and the soil surface was almost evenly covered by partially mineralized organic matter of grey-black color. In contrast, the plots affected by the high-severity fire had no remaining vegetation and the soil surface was mostly covered by white ash, suggesting an almost complete mineralization of organic matter. Soil sampling and analysis At each experimental plot, the soil sampling was carried out in three separate subplots in order to provide nine field replicates for each treatment (i.e. control, low-severity fire and highseverity fire). Soil sampling in each subplot included five soil cores, collected by a cylindrical plastic sampler (7 cm diameter and 5 cm height) from the soil surface down to a depth of 5 cm including ash, which were subsequently mixed together. As a previous study, carried out in a similar area of the same reserve, showed that plant species affect soil chemical– biological characteristics (Rutigliano et al. 2004), the soil was always collected under one plant species (P. angustifolia) that widely populates the study area. Soil samples were collected on 10 July, 25 September and 27 November 2000, and on 5 March and 2 July 2001, at 7, 84, 147, 245 and 364 days after burning. Int. J. Wildland Fire 357 For each soil sample (sieved through a 2-mm mesh and stored at 4◦ C prior to biological analyses), pH, organic carbon, catabolic response profiles, active fungal mycelia and microbial biomass carbon (Cmic ) were measured. Moreover, catabolic evenness and the fungal fraction of microbial carbon were calculated. The pH of air-dried soils was measured with a pH meter on a soil–water suspension (1 : 2.5 ratio). The organic matter was evaluated by loss-on-ignition at 550◦ C for 2 h and converted to organic carbon (Corg ), which is considered to be 58% of organic matter (Allen 1989). Catabolic response profiles were evaluated by measuring the short-term respiration response of fresh soil to the addition of simple organic compounds (Degens et al. 2000): 13 carboxylic acids (urocanic acid, succinic acid, citric acid, l-ascorbic acid, gluconic acid, malonic acid, dl-malic acid, α-ketoglutaric acid, fumaric acid, quinic acid, α-ketovaleric acid, pantothenic acid, α-ketobutyric acid), seven amino acids (l-lysine, l-histidine, l-glutamic acid, l-serine, l-arginine, l-asparagine, l-glutamine) and three carbohydrates (d-glucosamine, d-glucose, d-mannose). Soil respiration response was evaluated as CO2 evolved from fresh soil samples incubated in sealed vials for 4 h under standard conditions (25◦ C) after the addition of each substrate, using a gas chromatograph (Fisons GC 8000 series; Fisons Instruments, Milan, Italy) equipped as reported by Loftfield et al. (1997). The Cmic was evaluated by the substrate-induced respiration method (SIR; Degens et al. 2001), by gas chromatographic measuring of CO2 evolution from fresh soils (equivalent to 1 g of dry soil) after addition of 2 mL d-glucose solution (75 mm) and incubation in sealed vials for 4 h under standard conditions (25◦ C). Microbial biomass carbon was calculated from the glucose-induced respiration rate using the following equation (Degens et al. 2001): µg C g−1 soil = 50.4 × respiration(µL CO2 g−1 soil h−1 ). Active fungal mycelia were estimated using the membrane filter technique of Sundman and Sivelä (1978). Each fresh soil sample was dispersed in phosphate buffer (60 mm; pH 7.5) using a blender at 6000 rev. min−1 for 2 min and the soil suspension was strained through a membrane filter (0.45 µm mesh size); then the metabolically active mycelia on the filter surface were stained with fluorescein diacetate according to Söderström (1977). After clearing with immersion oil, the filter was examined at a magnification of ×400 and 20 microscopic fields were counted using an Axioskop MC 100 Spot Microscope (Carl Zeiss, Milan, Italy), equipped with an Hg lamp (HBO 50W). The total length of fungal mycelia was obtained using the intersection method (Olson 1950). The mass of active mycelia was calculated on the basis of the average values of cross section (9.3 µm2 ), density (1.1 g mL−1 ) and dry mass of the hyphae (15% of the wet mass) according to Berg and Söderström (1979). 358 R. D’Ascoli et al. Int. J. Wildland Fire Data analysis The catabolic evenness (E) was calculated from catabolic response profiles using the Simpson–Yule index: E = 1/
p2i , where pi is the respiration response to each substrate as a proportion of total respiration response to addition of all substrates (Magurran 1988). To obtain the fungal fraction of microbial carbon (i.e. the Cfung : Cmic ratio), the mycelium biomass was converted into carbon content (Cfung ), on the basis of mean values reported for C/N ratio (Killham 1994) and N content (Swift et al. 1979) in fungi, and then was expressed as a percentage of Cmic . Means and standard deviations reported in tables and figures were calculated from nine field replicates for each treatment (i.e. control, low-severity fire and high-severity fire). For each parameter the significance of differences between soils affected by different treatments was tested using one-way ANOVA, followed by the Student–Newman– Keuls test, using P < 0.05 as the significance threshold level (Sigma Stat 1.0; Systat Software, Erkrath, Germany). The linear correlations between different parameters were analysed using Pearson’s correlation coefficient (P < 0.05, n = 135; Sigma Stat 1.0). Results Functional diversity of the microbial community, assayed as catabolic response profiles (Fig. 1) and as catabolic evenness (Fig. 2), was different in soils affected by fire compared to the control only in the first week after disturbance. In particular, catabolic evenness showed significantly lower values in burned soils with differences between soils affected by different-severity fires (Fig. 2). In contrast, during the whole study period, respiration responses induced by each substrate were often lower in burned than in control soils, with the exception of respiration responses at 147 days after burning and at 364 days also for low-severity fire (Fig. 1). In addition, catabolic response profiles of soil affected by highseverity fire showed generally a higher percentage of changed responses, compared to control, than catabolic response profiles of soil burned with low-severity fire (Fig. 1). Active fungal mycelia (Table 1) were significantly lower in burned soils than in the control during the whole study period, except for soil affected by low-severity fire at 7 days, with a reduction compared to the control, from 16 to 45% after low-severity fire and from 36 to 55% after high-severity fire. A significant difference for active fungal mycelia was also found between soils affected by different-severity fire at 245 days after burning, with a reduction of 41% following the high-severity compared to low-severity fire. Unlike fungal mycelia, microbial carbon (Cmic , Table 1) was higher in burned soils at 7 and 84 days after fires, with an increase, compared to control, of 34 and 39% after low-severity fire and of 29 and 42% after high-severity fire, respectively. The fungal fraction of microbial carbon (Fig. 3), representing the relative importance of the fungal component within the soil microbial community, was changed in burned soils during the whole study period with a significant reduction, compared with unburned soil, of 19–57% after low-severity fire and of 37–61% after highseverity fire. The fungal fraction of microbial carbon was also significantly reduced (by 35%) at 245 days in soil affected by high-severity fire compared with soil burned by low-severity fire. In burned soils the Corg content also changed (Table 1), with significant increases, compared to the control, in soil affected by low-severity fire (from 20 to 39%) at 7, 147 and 364 days after fire, and in soil burned by high-severity fire (14%) only at 7 days after fire. Significant differences in organic carbon content between soils affected by differentseverity fires were also found at 7, 147 and 364 days after fires, with a reduction from 11 to 29% after high-severity fire compared with low-severity fire. A positive correlation between soil organic carbon and microbial biomass carbon (r = 0.385, P < 0.001) was found. An increase in the soil pH value was observed only at 7 days after high-severity fire, whereas a decrease in soil pH was found at 364 days after low- and high-severity fires, although all considered fluctuations in the pH values appeared very low. Negative correlations were found between soil pH and organic carbon, microbial biomass carbon and catabolic evenness (r = −0.180, P < 0.05; r = −0.633, P < 0.001; r = −0.542, P < 0.001, respectively) and positive correlations between pH and active fungal mycelia and fungal fraction of microbial carbon (r = 0.294, P < 0.01; r = 0.415, P < 0.001, respectively) were determined. No correlation was found between catabolic evenness and organic carbon, microbial biomass carbon, fungal mycelia and fungal fraction of microbial carbon. Discussion The microbial community showed a low resistance to fire disturbance, with resistance indicating the ability of the soil to withstand the immediate effects of perturbation (Griffiths et al. 2001), because all considered parameters were changed immediately after fire. In particular, changes in the catabolic response profiles were observed in the first week after fire and caused a reduction in catabolic evenness. A change in substrate utilisation pattern was also found by Pietikäinen et al. (2000) after short-term heating of dry forest humus (45–230◦ C), which was explained by the change in quality of organic matter, for the higher-temperature treatment. Degens et al. (2001) reported that the imposition of three stress types (i.e. decline in pH, increase in electrical conductivity, increase in Cu concentration) and two disturbance treatments (wet–dry or freeze–thaw cycles) caused reduction of catabolic evenness in crop soils with a low initial value for this parameter (i.e. 19.0 ± 0.19), but low or no effect on grazed soil with Microbial functional diversity in burned soils 150 Int. J. Wildland Fire Control Low-severity High-severity o 100 7 days after fire (July) 359 61% low 70% high * ** ** 50 ** ** ** ** 0 * ** ** ** ** ** ** ** 84 days after fire (September) ** 48% low 52% high 100 ** * ** Catabolic response (␮g CO2 g⫺1 dw h⫺1) 50 ** ** *o ** ** ** ** 0 ** 147 days after fire (November) **o ** 4% low 4% high 100 50 * 0 * 245 days after fire (March) 22% low 48% high 100 * 50 ** ** *o * 0 * * ** *o o 364 days after fire (July) 300 ** ** 4% low 22% high 200 100 ** 50 *o 0 *o *o *o o e e e e e e e d e e d d d d id d cid cid aci aci aci aci acid sin din acid erin inin gin min min cos nos cid aci ac aci a a g ara ta sa Glu an c -S c nic ric a bic nic nic lic a ric a ric nic ric nic ric -Ly isti i r i L L A lu o DH am M an ci Cit or co alo Ma uta ma ui ale the uty L- A s p - G l u c t LQ ov to c lu M Dl Fu oc Suc b L lu Lt -G DL tog As G G n eto Ur e D a Le L K P K K cid cid Fig. 1. Functional diversity of the microbial community, as catabolic response profiles after addition of different organic compounds, in burned soils following low- and high-severity fires and in unburned control soil, at different time after fire. Percentage of changed responses in burned soils (compared to control) is reported in each graph in the top right-hand corner. Standard error is given in the bars. Significant differences (P < 0.05) between burned soils and control are indicated by asterisks, significant differences between soils burned with different-severity fires are indicated by circles. higher initial catabolic evenness (i.e. 21.4 ± 0.17). Therefore, we can infer that, after fire disturbance, the reduction of catabolic evenness may be due to a low value of catabolic evenness in soil of the study area (i.e. 14.01 ± 0.25) indicating that catabolic activity was not homogeneously distributed between different functions. As from 3 months after fire, soils recovered their functional diversity, that is the normal balance between the considered catabolic functions. This effect may be explained by the increase in organic and microbial carbon immediately after fire that may have a protective 360 R. D’Ascoli et al. Int. J. Wildland Fire function for the microbial community. In fact, Degens et al. (2001) hypothesized that high values of some soil properties, such as organic carbon content, cation exchange capacity and microbial biomass, may also increase the resistance of soil catabolic evenness, because of the great protective effect of these properties on the microbial community, favouring microbial recolonisation after stress or disturbance. Moreover, a reduction in respiration responses induced by simple organic compounds was observed in burned compared to control soils during the whole study period, except at 147 days after fire, which corresponds to the rainy season (i.e. autumn) for the Mediterranean region. We can hypothesize that, under non-limiting water conditions typical of the autumn season, soil microorganisms showed complete recovery of their activity whereas, in the other seasons, they showed lower activity 15 Control Low-severity fire High-severity fire 20 Control Low-severity fire High-severity fire Cfung (%Cmic) Catabolic evenness 25 15 * o * 10 10 * * *o 5 5 * * * * * 7 (Jul) 84 (Sep) * 0 0 7 (Jul) 84 147 245 (Sep) (Nov) (Mar) Days after fire 364 (Jul) 147 (Nov) 245 (Mar) 364 (Jul) Days after fire Fig. 2. Catabolic evenness in soils affected by low- and high-severity fires and in control soil. Standard error is given in the bars. For each sampling date, asterisks indicate significant (P < 0.05) differences between burned and unburned soils, circles indicate significant differences between soils burned with different-severity fires. Fig. 3. Fungal fraction of microbial carbon, Cfung (%Cmic ), in soils affected by low- and high-severity fires and in control soil. Standard error is given in the bars. For each sampling date, significant (P < 0.05) differences between burned and unburned soils are indicated by asterisks, significant differences between soils burned with different-severity fires are indicated by circles. Table 1. Variations in chemical and biological parameters after fires Mean values (± standard error) of pH, organic carbon (Corg ), microbial biomass carbon (Cmic ) and active fungal mycelia 7 days after fire Control Low-severity fire High-severity fire 84 days after fire Control Low-severity fire High-severity fire 147 days after fire Control Low-severity fire High-severity fire 245 days after fire Control Low-severity fire High-severity fire 364 days after fire Control Low-severity fire High-severity fire A Significant B Significant pH Corg (%) Cmic (mg g−1 dry weight) Active fungal mycelia (mg g−1 dry weight) 7.3 (± 0.0) 7.2 (± 0.1) 7.5 (± 0.0)A,B 4.72 (± 0.22) 6.03 (± 0.12)A 5.36 (± 0.16)A,B 0.840 (± 0.074) 1.126 (± 0.093)A 1.087 (± 0.073)A 0.073 (± 0.006) 0.061 (± 0.006) 0.044 (± 0.007)A 7.2 (± 0.1) 7.2 (± 0.1) 7.1 (± 0.1) 4.80 (± 0.27) 5.77 (± 0.32) 5.41 (± 0.20) 0.907 (± 0.083) 1.261 (± 0.115)A 1.289 (± 0.060)A 0.113 (± 0.014) 0.062 (± 0.005)A 0.056 (± 0.006)A 7.8 (± 0.0) 7.8 (± 0.0) 7.8 (± 0.0) 4.48 (± 0.50) 6.22 (± 0.39)A 5.21 (± 0.39)B 0.598 (± 0.042) 0.661 (± 0.055) 0.604 (± 0.024) 0.160 (± 0.009) 0.102 (± 0.006)A 0.102 (± 0.005)A 7.6 (± 0.1) 7.5 (± 0.1) 7.6 (± 0.0) 4.97 (± 0.71) 6.50 (± 0.41) 4.74 (± 0.32) 0.794 (± 0.102) 0.639 (± 0.039) 0.546 (± 0.054)A 0.173 (± 0.013) 0.132 (± 0.014)A 0.078 (± 0.006)A,B 7.9 (± 0.0) 7.6 (± 0.0)A 7.8 (± 0.0)A,B 5.36 (± 0.69) 7.25 (± 0.48)A 5.13 (± 0.38)B 0.749 (± 0.075) 0.978 (± 0.110) 0.805 (± 0.103) 0.126 (± 0.010) 0.076 (± 0.004)A 0.077 (± 0.004)A (P < 0.05) differences between burned and unburned soils. (P < 0.05) differences between soils burned with different-severity fires. Microbial functional diversity in burned soils in burned soils than in unburned soils, probably because of an indirect effect of fire on soil. In fact, the reduction of plant cover in burned soils may cause a more marked fluctuation of microclimate resulting in more stress for the microbial community in the dry season. Díaz-Raviña et al. (1996) explained the prolonged reduced bacterial activity in heated soils with the presence of toxic substances, as shown by the inhibitory effect on bacterial activity of water extract from heated soils. According to the reduction in respiration responses, active fungal mycelia and the fungal fraction of microbial carbon were reduced in burned soils during the whole study period, indicating a persistent alteration of the microbial community after burning. Similarly, Pietikäinen and Fritze (1993) reported that fungal growth in burned soil did not reach the values measured in the unburned soil within the first 3 years after fire. Also, other authors (e.g. Carballas et al. 1993; Vásquez et al. 1993) emphasized the negative effect of wildfire on fungal populations. Fungi are more sensitive than bacteria to the increased soil temperature during fire (Rundel 1981). The heat-tolerance limit reported by Dunn and DeBano (1977) in chaparral soils is higher for heterotrophic bacteria (210◦ C) than for fungi (155◦ C). Pietikäinen et al. (2005) have shown that fungal and bacterial growth rates have optimum temperatures around 25–30◦ C: at higher temperatures lower growth rates are found, although this decrease is more drastic for fungi than for bacteria, resulting in an increase in the ratio of bacterial to fungal growth rate at higher temperatures. In our study, the decrease in fungi in the first week after fire is probably due to direct disturbance by fire (i.e. the increase in soil temperature during the fire), whereas the persistence of a low fungal growth, in the following study period, may be explained by fungi sensitivity to stress conditions consisting of more marked variations in soil moisture and temperature, resulting from the reduction of plant cover after burning (Rutigliano et al. 2002b). Fungi may also be sensitive to the killing of plants by fire causing a cessation of root growth and exudation (Bääth 1980). Other authors explained the fungal reduction in burned soil with the release of chemicals inhibiting fungal growth (Widden and Parkinson 1975). During the first week after fire, for soils burned at high fire severity, an effect of the increase in soil pH on fungal growth and fungal : bacterial ratio cannot be excluded. The increase in soil pH, typically found after fire, can affect the soil microbial community because it favours bacterial over fungal population growth (Rundel 1981). The reduction in fungal biomass in the studied soils may indicate a reduction in decomposing activity specifically linked to them, such as lignin and cellulose degradation. In fact it is well known that fungi are the main decomposers of plant material (Anderson 1981). Microbial biomass carbon increased until 3 months after fire. This increase probably reflects an increase in microbial community growth of the bacterial component only, because fungi were reduced. The bacterial increase may be due both Int. J. Wildland Fire 361 to adaptation to fire (the Mediterranean maquis being a fire-prone ecosystem) and to an increase in soil organic matter in burned soils on the basis that microbial carbon was positively correlated with organic carbon. The increase in organic carbon in soil burned at low fire severity may be explained by the input of partially mineralised plant biomass, as shown by grey-black residues left on the soil by fire. Also, an input to the organic carbon pool derived from soil fauna larger than 2 mm (excluded by sieving in control soil) and killed but not completely mineralised by the fire, cannot be excluded. McKee (1982) observed an increase in organic carbon in the soil surface and explained it by the incorporation of charcoal and partially burned organic matter into the mineral soil and, in some cases, by the increase in the presence of N-fixing species following burning. In our studied soil affected by high fire severity, the increase in organic carbon, which was lower than in soil affected by low-severity fire and found only in the first week after fire, was probably due mainly to an input of material other than partially mineralized plant biomass, because the high-severity burning produced more marked mineralisation of biomass, as demonstrated by the presence of white ash on soil after burning. The fire impact appeared more marked after high-severity fire than after low-severity fire, as suggested by a higher reduction of catabolic evenness and a higher percentage of change in specific activities (compared to control). A more marked effect of high- compared to low-severity fires was also reported by Pietikäinen and Fritze (1993) for fungal mycelium length. Conclusions In the Mediterranean maquis area of the Castel Volturno Nature Reserve, fire caused a change in soil microbial community, with generally more marked effects after highseverity fire than after low-severity fire. The alteration of catabolic response profiles and the reduction of catabolic evenness in burned soils were observed only in the first week after burning, indicating that in these soils the microbial community was able to quickly recover its functional diversity. Nevertheless in burned soils, respiration responses to the addition of simple organic compounds were generally reduced during the whole study period, with the exception of the rainy season, indicating a reduction of specific activities. Moreover, active fungal mycelia were reduced in burned soil until 1 year after the burning, while the increase in soil organic carbon generally observed during the first year after burning promoted the growth of soil total microbial biomass within the first 3 months. The different response to fire of fungi, compared with bacteria, caused a change in microbial community composition. In fact, the fungal fraction of microbial carbon was reduced in burned soils until 1 year after fires, indicating that the structural alteration of microbial community persists for a long time following low- and high-severity fires. The results suggest that burned soils of Mediterranean 362 Int. J. Wildland Fire maquis can quickly recover their functional diversity following fire, despite the persistent reduction of the activity of the microbial community and its change in structure. Acknowledgements This work was supported by Ministero dell’Istruzione dell’Università e della Ricerca of Italy. The authors thank the Forest Service of the Castel Volturno Nature Reserve for technical assistance to carry out the experimental fires. References Agrawal AK (1990) Floristic composition and phenology of temperate grasslands of Western Himalaya as affected by scraping, fire and heavy grazing. Vegetatio 88, 177–187. doi:10.1007/BF00044834 Allen SE (1989) ‘Chemical analysis of ecological materials.’ (Blackwell Scientific Publications: Oxford) Anderson JM (1981) ‘Ecology for environmental sciences: biosphere, ecosystems and man.’ (Edward Arnold: London) Bääth E (1980) Soil fungal biomass after clear-cutting of a pine forest in Central Sweden. Soil Biology & Biochemistry 12, 495–500. doi:10.1016/0038-0717(80)90086-3 Beer T, Gill AM, Moore PHR (1988) Australian bushfire danger under changing climate regimes. In ‘Greenhouse: planning for climate change’. (Ed. GI Pearman) pp. 421–427. (CSIRO: East Melbourne) Bengtsson J (1998) Which species? What kind of diversity? Which ecosystem function? Some problems in studies of relations between biodiversity and ecosystem function. Applied Soil Ecology 10, 191–199. doi:10.1016/S0929-1393(98)00120-6 Berg B, Söderström B (1979) Fungal biomass and nitrogen in decomposing Scots pine needle litter. Soil Biology & Biochemistry 11, 339–341. doi:10.1016/0038-0717(79)90045-2 Boo RM, Pelaez DV, Bunting SC, Mayor MD, Elia OR (1997) Effect of fire on woody species in Central semi-arid Argentina. Journal of Arid Environments 35, 87–94. doi:10.1006/JARE.1995.0135 Booyesen P de V, Tainton NN (Eds) (1984) ‘Ecological effects of fire in South African ecosystems.’ (Springer-Verlag: Berlin) Brussaard L, Behanpelletier VM, Bignell DE, Brown VK, Didden W, et al. (1997) Biodiversity and ecosystem functioning in soil. Ambio 26, 563–570. Carballas M, Acea MJ, Cabaneiro A, Trasar C, Villar MC, et al. (1993) Organic matter, nitrogen, phosphorus and microbial population evolution in forest humiferous acid soils after wildfires. In ‘Fire in Mediterranean ecosystems’. (Eds L Trabaud, R Prodon) pp. 379–385. Ecosystems research report no. 5. (Commission of the European Communities: Brussels) De Luis M, Baeza MJ, Raventós J, González-Hidalgo JC (2004) Fuel characteristics and fire behaviour in mature Mediterranean gorse shrublands. International Journal of Wildland Fire 13, 79–87. doi:10.1071/WF03005 Degens BP (1999) Catabolic response profiles differ between microorganisms grown in soils. Soil Biology & Biochemistry 31, 475–477. doi:10.1016/S0038-0717(98)00133-3 Degens BP, Schipper LA, Sparling GP, Vojvodic-Vukovic M (2000) Decreases in organic C reserves in soil can reduce the catabolic diversity of soil microbial communities. Soil Biology & Biochemistry 32, 189–196. doi:10.1016/S0038-0717(99)00141-8 Degens BP, Schipper LA, Sparling GP, Duncan LC (2001) Is the microbial community in a soil with reduced catabolic diversity less resistant to stress or disturbance? Soil Biology & Biochemistry 33, 1143–1153. doi:10.1016/S0038-0717(01)00018-9 Díaz-Raviña M, Prieto A, Bääth E (1996) Bacterial activity in a forest soil after soil heating and organic amendments measured by R. D’Ascoli et al. the thymidine and leucine incorporation techniques. Soil Biology & Biochemistry 28, 419–426. doi:10.1016/0038-0717(95)00156-5 di Gennaro A (2002) ‘I sistemi di terre della Campania.’ (Edizioni S.EL.CA.: Firenze) Dumontet S, Dinel H, Scopa A, Mazzatura A, Saracino A (1996) Post-fire soil microbial biomass and nutrient content of a pine forest soil from a dunal mediterranean environment. Soil Biology & Biochemistry 28, 1467–1475. doi:10.1016/S0038-0717(96)00160-5 Dunn PH, DeBano LF (1977) Fire’s effects on the biological properties of chaparral soils. In ‘Proceedings of symposium on the environmental consequences of fire and fuel management in Mediterranean ecosystems’. USDA Forest Service General Technical Report WO-3. pp. 75–84. (Washington, DC) Esposito A, Strumia S, Buonanno M, Castaldo-Cobianchi R, Mazzoleni S (1998) Analysis of bryophyte dynamics after fires of pine woodlands and Mediterranean macchia, southern Italy. In ‘Fire management and landscape ecology’. (Ed. L Trabaud) pp. 77–85. (International Association of Wildland Fire: Fairfield, WA) Esposito A, Mazzoleni S, Strumia S (1999) Post-fire bryophyte dynamics in Mediterranean vegetation. Journal of Vegetation Science 10, 261–268. FAO (1998) World reference base for soil resources. In ‘World soil resources’. Report No. 84. (FAO: Rome) Flannigan MD, Van Wagner CE (1991) Climate change and wildfire in Canada. Canadian Journal of Forest Research 21, 66–72. Fritze H, Pennanen T, Pietikäinen J (1993) Recovery of soil microbial biomass and activity from prescribed burning. Canadian Journal of Forest Research 23, 1286–1290. Gill AM, Groves RH, Noble IR (Eds) (1981) ‘Fire and the Australian biota.’ (Australian Academy of Science: Canberra) Giller KE, Beare MH, Lavalle P, Izac AMN, Swift MJ (1997) Agricultural intensification, soil biodiversity and agroecosystem function. Applied Soil Ecology 6, 3–16. doi:10.1016/S0929-1393(96)00149-7 Griffiths BS, Bonkowski M, Roy J, Ritz K (2001) Functional stability, substrate utilisation and biological indicators of soils following environmental impacts. Applied Soil Ecology 16, 49–61. doi:10.1016/S0929-1393(00)00081-0 Hobbs RJ, Atkins L (1990) Fire-related dynamics of a Banksia woodland in south-western Australia. Australian Journal of Botany 38, 97–110. Iovieno P, Alfani A, Rutigliano FA, Virzo De Santo A (1996) Inquinamento urbano. 3. Morfologia delle ife fungine in relazione al contenuto di sostanza organica e di elementi in traccia nel suolo. In ‘Atti del VII Congresso Nazionale della Società Italiana di Ecologia, Napoli, Italy, S.It.E. Atti 17’. (Eds Società Italiana di Ecologia) pp. 733–736. (Giannini & Figli: Napoli) Killham K (1994) ‘Soil ecology.’ (Cambridge University Press: Cambridge) Laterra P (1997) Post burn recovery in the flooding pampa: impact of an invasive legume. Journal of Range Management 50, 274–277. Le Coz J (1990) ‘Espaces méditerranéens et dynamiques agraires: état territorial et communautés rurales. Option méditerranéens Serie B 2.’ (Ciheam-Unesco/Mab: Paris) Loftfield N, Flessa H, Augustin J, Beese F (1997) Automated gas chromatographic system for rapid analysis of the atmospheric trace gases methane, carbon dioxide and nitrous oxide. Journal of Environmental Quality 26, 560–564. Magurran AE (1988) ‘Ecological diversity and its measurement.’ (Croom Helm: London) McKee WH (1982) ‘Changes in soil fertility following prescribed burning on coastal plain pine sites.’ USDA Forest Service, Southeastern Forest Experiment Station Research Paper SE-234. Molina MJ, Llinares JV (1998) Effects of fire intensity on the soil properties related to structure: organic matter, aggregate stability and water retention capacity. In ‘Fire management and landscape ecology’. Microbial functional diversity in burned soils Int. J. Wildland Fire (Ed. L Trabaud) pp. 35–50. (International Association of Wildland Fire: Fairfield, WA) Molina MJ, Llinares JV (2001) Temperature–time curves at the soil surface in maquis summer fires. International Journal of Wildland Fire 10, 45–52. doi:10.1071/WF01001 Moreno JM, Oechel WC (1994) Fire intensity as a determinant factor of postfire plant recovery in southern California chaparral. In ‘The role of fire in Mediterranean-type ecosystems’. (Eds JM Moreno, WC Oechel) Ecological Studies 107, pp. 26–45. (Springer-Verlag: New York) Naveh Z (1975) The evolutionary significance of fire in the Mediterranean region. Vegetatio 29, 199–208. Nordgren A, Bååth E, Soderstrom B (1983) Microfungi and microbial activity along a heavy metal gradient. Applied and Environmental Microbiology 45, 1829–1837. Olson FCW (1950) Quantitative estimates of filamentous algae. Transactions of the American Microscopical Society 69, 272–279. Olson JS (1981) Carbon balance in relation to fire regimes. In ‘Fire regimes and ecosystem properties’. (Eds HA Mooney, TM Bonnicksen, NL Chrinstensen, JE Lotan, WA Reiners) pp. 327–378. USDA Forest Service General Technical Report WO-26. (Washington, DC) Pietikäinen J, Fritze H (1993) Microbial biomass and activity in the humus layer following burning: short term effects of two different fires. Canadian Journal of Forest Research 23, 1275–1285. Pietikäinen J, Fritze H (1995) Clear-cutting and prescribed burning in coniferous forest: comparison of effects on soil fungal and total microbial biomass, respiration activity and nitrification. Soil Biology & Biochemistry 27, 101–109. doi:10.1016/0038-0717(94)00125-K Pietikäinen J, Hiukka R, Fritze H (2000) Does short-term heating of forest humus change its properties as a substrate for microbes? Soil Biology & Biochemistry 32, 277–288. doi:10.1016/S00380717(99)00164-9 Pietikäinen J, Pettersson M, Bääth E (2005) Comparison of temperature effects on soil respiration and bacterial and fungal growth rates. FEMS Microbiology Ecology 52, 49–58. doi:10.1016/ J.FEMSEC.2004.10.002 Prodon R, Fons R, Athias-Binche F (1987) The impact of fire on animal communities in Mediterranean area. In ‘The role of fire in ecological systems’. (Ed. LTrabaud) pp. 121–157. (SPBAcademic: The Hague) Rahkonen J, Pietikäinen J, Jokela H (1999) The effects of flame weeding on soil microbial biomass. Biological Agriculture and Horticulture 16, 363–368. Rundel PW (1981) Fire as an ecological factor. In ‘Physiological plant ecology. I. Responses to the physical environment’. (Eds OL Lange, PS Nobel, CB Osmond, H Ziegler) Encyclopedia of Plant Physiology, New Series, Vol. 12A, pp. 501–538. (Springer-Verlag: Berlin) Rutigliano FA, Virzo De Santo A, Alfani A, Esposito A (1995) Attività metabolica di suoli campani in funzione degli incendi. In ‘Atti del VI Congresso Nazionale della Società Italiana di Ecologia, Venezia, Italy, S.It.E. Atti 16’. (Eds Società Italiana di Ecologia) pp. 535–538. (Edizioni Zara: Parma) Rutigliano FA, FierroAR, De Pascale RA, De MarcoA,Virzo De SantoA (2002a) Role of fire on soil organic matter turnover and microbial activity in a Mediterranean burned area. In ‘Soil mineral–organic 363 matter–microorganism interactions and ecosystem health’. (Eds A Violante, PM Huang, J-M Bollag, L Gianfreda) Developments in soil science 28B, pp. 205–215. (Elsevier Science: Amsterdam) Rutigliano FA, D’Ascoli R, De Marco A, Virzo De Santo A (2002b) Soil microbial community as influenced by experimental fires of different intensities. In ‘Fire and biological processes’. (Eds L Trabaud, R Prodon) pp. 137–149. (Backhuys Publishers: Leiden) Rutigliano FA, D’Ascoli R, Virzo De Santo A (2004) Soil microbial metabolism and nutrient status in a Mediterranean area as affected by plant cover. Soil Biology & Biochemistry 36, 1719–1729. doi:10.1016/J.SOILBIO.2004.04.029 Schaefer JA (1993) Spatial patterns in Taiga plant communities following fire. Canadian Journal of Botany 71, 1568–1573. Söderström B (1977) Vital staining of fungi in pure cultures and in soil with fluorescein-diacetate. Soil Biology & Biochemistry 11, 237–246. Sundman V, Sivelä S (1978) A comment on the membrane filter technique for estimation of length of fungal hyphae in soil. Soil Biology & Biochemistry 10, 399–401. doi:10.1016/0038-0717(78)90065-2 Swift MJ, Heal OW, Anderson JM (1979) ‘Decomposition in terrestrial ecosystems.’ (Blackwell Scientific Publications: Oxford) Trabaud L, Grandjanny M (2002) Post-fire reconstitution of the flowering phenology in Mediterranean shrubland plants. In ‘Fire and biological processes’. (Eds L Trabaud, R Prodon) pp. 99–113. (Backhuys Publishers: Leiden) Trabaud L, Lepart J (1980) Diversity and stability in garriga ecosystems after fire. Vegetatio 43, 49–57. doi:10.1007/BF00121017 Torn MS, Fried JS (1992) Predicting the impacts of global warming on wildland fire. Climatic Change 21, 257–274. doi:10.1007/ BF00139726 Trevors JT (1998) Bacterial biodiversity in soil with an emphasis on chemically contaminated soils. Water, Air, and Soil Pollution 101, 45–67. doi:10.1023/A:1004953404594 van Veen JA, Heijnen CE (1994) The fate and activity of microorganisms introduced into soil. In ‘Soil biota: management in sustainable farming systems’. (Eds CE Pankhurst, BM Double, VVSR Gupta, PR Grace) pp. 63–71. (CSIRO: Adelaide) Vásquez FJ, Acea MJ, Carballas T (1993) Soil microbial population after wildfire. FEMS Microbial Ecology 13, 93–104. doi:10.1016/01686496(93)90027-5 Whittle CA, Duchesne LC, Needham T (1997) The impact of broadcast burning and fire severity on species composition and abundance of surface vegetation in a Jack pine (Pinus banksiana) clear-cut. Forest Ecology and Management 94, 141–148. doi:10.1016/S03781127(96)03969-2 Widden P, Parkinson D (1975) The effects of a forest fire on soil microfungi. Soil Biology & Biochemistry 7, 125–138. doi:10.1016/00380717(75)90010-3 Wright HA, Bailey AW (1982) ‘Fire ecology. United States and Southern Canada.’ (John Wiley & Sons: New York) Zak JC, Willig MR, Moorhead DL, Widman HG (1994) Functional diversity of microbial communities: a quantitative approach. Soil Biology & Biochemistry 26, 1101–1108. doi:10.1016/00380717(94)90131-7 http://www.publish.csiro.au/journals/ijwf