The Specific and Total CO2 Emission Activity of Wood-Decaying Fungi and Their Response to Increases in Temperature

The CO2 emission activity of xylotrophic fungi responding to an increase in temperature in the range of 10–30 °C with pure dikaryotic cultures of Fomes fomentarius s. str., F. inzengae, Fomitopsis betulina, F. pinicola, and Phellinus igniarius was analyzed. Emission activity was assessed by the difference in CO2 concentration in 0.5 L exposure chambers with Petri dishes with mycelium growing on agar at the beginning of exposure and an hour later using a Gasmet DX-4030 FTIR spectrometer (Gasmet Technologies Oy, Finland), error measurements ±50 ppm. Specific (μg CO2/cm2/h) and total (μg CO2/h) emission activity and its relationship with temperature and size (area) of the mycelium were assessed. It is shown that in the range of 10–30 °C, the specific and total CO2 emission activity of the mycelium is closely and positively related to temperature. Specific emission, which is an indicator of the respiratory activity of the mycelium, does not depend on its size; its only driver is temperature, the relationship with which is linear: an increase in temperature by 10 °C causes an increase in the specific emission activity of the mycelium by 1.7 times. The total CO2 emission activity, which is an indicator of the total amount of CO2 emitted, is directly proportional to the specific emission activity and the size of the mycelium. In the range of 10–30 °C, an increase in temperature causes an almost equal increase in both the specific emission activity of the mycelium (Q10 1.7) and its growth (Q10 1.5) and causes an exponential increase in the total emission of CO2. This must be taken into account when predicting CO2 emissions from woody debris under climate change, as it could potentially contribute to accelerating climate change.


Introduction
Climate change, its causes, and environmental consequences are some of the most discussed problems in ecology, as well as in works on the ecology of woody debris and its decomposition [1][2][3][4].This is far from accidental, since woody debris is a globally significant natural reservoir of carbon; in Russian forests alone, its reserves amount to about 5.5 Gt.This huge mass of woody debris is at various stages of its biological decomposition-a large-scale process specific to forest ecosystems, during which annually about 214 Mt C-CO 2 is emitted into the atmosphere from Russian forests.This makes debris the second largest natural source of carbon dioxide after soil [1,5].
The main role in the decomposition of debris in forests of temperate latitudes is played by xylotrophic fungi-Basidiomycota and Agaricomycetes [6][7][8][9].This is, perhaps, the only group of organisms in the modern biosphere that has a unique set of interconnected ecological and physiological adaptations to the woody habitat and is capable of decomposing the lignocellulosic complex of wood [10].They determine the main parameters of CO 2 emission activity of woody debris, and this makes them one of the globally significant regulators of the gas composition of the atmosphere [11], which is a factor of climate stability and change [4].
One of the key questions arising from climate change relates to future carbon dynamics, which largely depend on the temperature sensitivity of decomposition processes.They play an important role in the global carbon cycle and, through feedback, can potentially influence climate change [4,12,13].All currently available data clearly indicate a close connection between the CO 2 emission activity of xylotrophic fungi, wood debris, and temperature [2][3][4][14][15][16][17]. In particular, in one of our recent works [18], we showed that an increase in temperature from 20 to 30 • C has a nonadditive, possibly synergistic effect on the CO 2 emission activity of xylotrophic fungi, causing its exponential growth.
Considering the role of xylotrophic fungi as factors of stability and climate change, this phenomenon undoubtedly requires most careful study and, above all, this concerns the temperature response of CO 2 emission activity of xylotrophic fungi to an increase in temperature.This determined the purpose of this work-an analysis of the relationships between the CO 2 emission activity of xylotrophic fungi and temperature, testing the hypothesis of an exponential increase in CO 2 emission with increasing temperature in the range of 10-30 • C, which is relevant for temperate latitudes.

Materials and Methods
A study of the temperature response of xylotrophic fungi CO 2 emission activity was carried out on dikaryotic mycelia of Fomes fomentarius (L.) Fr., F. inzengae (Ces.and De Not.) Cooke, Fomitopsis betulina (Bull.)B.K. Cui, M. L. Han and Y.C. Dai, F. pinicola (Sw.)P. Karst., and Phellinus igniarius (L.) Quél.growing on wort agar (Figure 1).The CO2 content in the chambers was measured using a Gasmet DX-4030 FTIR spectrometer (Gasmet Technologies Oy, Finland) with an accuracy of ±50 ppm.The emission activity of the mycelium was assessed by the difference in CO2 concentration in the chambers at the beginning of the exposure and at the end and was calculated in μg of CO2, taking into account the volume of the exposure chambers and Petri dishes and the exposure duration.
Specific CO2 emission was calculated in μg CO2/cm 2 /h by Equation ( 1): Dikaryotic cultures were isolated from basidiocarps of the corresponding species of fungi using traditional methods [19] and wort (4%)-agar (2%) as a nutrient medium (MA).The use of pure cultures allows one to solve one of the most difficult problems in the study of xylotrophic fungi-the assessment of mycelium biomass in wood.When mycelium develops on artificial nutrient media, an indicator of its biomass can simply be assessed by the area it occupies in Petri dishes.
The species identification of basidiocarps was determined on anatomical and morphological characteristics [20], and their species names are given according to Index Fungorum [21].F. fomentarius strains were typed using ITS region sequencing; according to phylogenetic analysis, strains collected on Populus L. belong to F. inzengae and on Betula L. belong to F. fomentarius sensu stricto-two cryptic taxa [22][23][24].
The analysis scheme was as follows.Petri dishes (9 cm in diameter, 3 for each strain) were inoculated with a piece of agar (about 5 mm) with the mycelium of the fungus being studied and kept for several days at +25 • C. When the mycelium began to grow around the inoculum, its border was marked by felt-tip pen on the underside of the Petri dishes, and dishes were placed in open exposure chambers with a volume of 0.5 L and placed in a thermostat at +10 • C for 2 h.Then, the chambers were sealed, their CO 2 content was measured, and they were placed in a thermostat at +10 • C for one hour.At the end of the exposure, CO 2 measurements in the chambers were made again, after which they were opened and left with closed Petri dishes inside for a day in a thermostat at +10 • C.After 24 h, the size of the mycelia in the Petri dishes was measured, the borders were marked, the chambers were closed, CO 2 was measured, and they were placed in a thermostat at +10 • C for an hour.At the end of the exposure, the CO 2 content in the chambers was measured again.According to the same scheme, the growth of mycelium and its emission activity were assessed at +20 The CO 2 content in the chambers was measured using a Gasmet DX-4030 FTIR spectrometer (Gasmet Technologies Oy, Finland) with an accuracy of ±50 ppm.The emission activity of the mycelium was assessed by the difference in CO 2 concentration in the chambers at the beginning of the exposure and at the end and was calculated in µg of CO 2, taking into account the volume of the exposure chambers and Petri dishes and the exposure duration.
Specific CO 2 emission was calculated in µg CO 2 /cm 2 /h by Equation ( 1): The total CO 2 emission, or the total amount of carbon dioxide emitted by the mycelium, was calculated in µg CO 2 /h by Equation ( 2): where SEA is the specific CO 2 emission, TEA is the total CO 2 emission, ∆CO 2 is the amount of CO 2 released by the mycelium during exposure (ppm/h), V 1 is the chamber volume (l), V 2 is the sample volume (l), Vm is the molar volume (22.4 l/mol), M is the molar mass of CO 2 (44 g/mol), S is the area occupied by mycelium (cm 2 ), and T is the temperature in Kelvin (K).
The temperature coefficient (Q 10 ) of specific CO 2 emission, showing the multiplicity of its change with a temperature increase of 10 • C, was calculated by Equation (3): where Q 10 SEA is the temperature coefficient of specific emission, SEA 1 is the specific emission at 10 and 20 • C, and SEA 2 at 20 and 30 • C, respectively.The temperature dependence of mycelium growth was assessed by the increase in the area it occupied on MA during the day (cm 2 /day) and by the temperature coefficient (Q 10 ) of growth, calculated by the similar Equation ( 4): where Q 10 SM is the temperature coefficient of growth, V 1 is the intensity of mycelium growth (cm 2 /day) at 10 and 20 • C, and V 2 is the intensity of mycelium growth at 20 and 30 • C, respectively.Statistical data processing was performed using the Statistica 10.0 program (StatSoft Inc., Tulsa, OK, USA).Arithmetic means (m) are given with standard errors (SE).The Pearson correlation coefficient (r) was used to characterize the relationships between variables.Student's t-test was used for pairwise comparisons; one-way analysis of variance (ANOVA) was used for multiple comparisons of means.The correspondence of the CO 2 emission activity temperature dynamics with a linear (SEA = a + b × t, where t is the temperature in degrees Celsius) or exponential (TEA = a × exp(b × t)) regression model was assessed on coefficient of determination or R 2 -a statistical measure correspondence of regression line to the actual data.When describing the results of statistical evaluation, the values of the corresponding criteria and their significance are given.

Results
Figure 2 shows the dynamics of CO 2 emission activity of five species of dikaryotic mycelium at MA in the range of 10-30 • C. It is seen that the temperature dynamics of total (TEA) and specific (SEA) CO 2 emission activity are significantly different: SEA is linear (determination coefficient 0.91-0.98),and TEA is exponential (determination coefficient 0.94-0.99).Tables 1-3 show that the temperature raise from 10 to 20 • C increased the specific CO 2 emission activity of mycelium from 1.3 (F.betulina) to 2.1 (F pinicola, collected on Picea), on average 1.8 times.An increase in temperature from 20 to 30 • C enhances SEA by 1.2 (F.fomentarius s. str.)-1.9 (F.pinicola, collected on Picea) on average 1.6 times.In other words, the temperature coefficient of the specific CO 2 emission activity of the mycelium of the studied group of xylotrophic fungi ranges from 1.6 to 1.8.An increase in temperature from 10 to 30 • C (3 times) causes a corresponding increase in SEA-2.9 times.At 35 • C, SEA decreases in some of the analyzed fungi, while in F. betulina and F pinicola (collected on Betula) it remains at the same level as at 30 • C.  The specific CO 2 emission activity of the mycelium does not show any relationship connection with its size, in our case, with its area.Thus, in the F. betulina strain at 10 • C SEA of the same level (18-19 µg CO 2 /cm 2 /h) for mycelium with an area of 10 and 13 cm 2 , and at 20 • C, it is equal to 24-25 µg CO 2 /cm 2 /h for mycelium with an area of 13 and 21 cm 2 (Table 1).In F. pinicola (strain collected on Picea) at 10 • C, SEA equal to 19 µg CO 2 /cm 2 /h is recorded in mycelium with an area of 11 and 14 cm 2 , as well as in the strain collected on Betula 23 µg CO 2 /cm 2 /h in mycelium of 13 and 18 cm 2 .
The same is observed at 30 • C: in the strain collected on Betula, the mycelium of 29 cm 2 and 38 cm 2 has SEA equal to 73 µg CO 2 /cm 2 /h, as well as in the strain collected on Picea 73-74 µg CO 2 /cm 2 /h in the mycelium with an area of 27 cm 2 and 35 cm 2 (Table 2).There is also no relationship between SEA and mycelium area in the F. fomentarius s. str.and F. inzengae strains (Table 3).
The response of total CO 2 emission activity to an increase in temperature is more pronounced than in the case of specific activity.If, with an increase in temperature from 10 to 20 • C, SEA, as noted, increases by 1.3-2.1 times on average 1.8 times only, then TEA increases by 3 (F.betulina)-6 (F.fomentarius s. str.) times, on average 4 times.When the temperature increases from 20 to 30 • C, TEA increases by 2 (F.betulina)-4 (F.fomentarius s. str., F. pinicola), on average 3 times, while SEA 1.6 times.An increase in temperature from 10 to 30 • C enhances the TEA of the mycelium of F. betulina and Ph.igniarius by 6, F. pinicola by 10, F. inzengae by 13, and F. fomentarius s. str.by 21 times-on average 11 times.At the same time, as noted above, SEA will increase by 2.9 times.Like SEA, TEA reaches its maximum at 30 • C; at 35 • C, it decreases or remains at the same level as at 30 • C. TEA varies depending on the fungus species: the highest (2000-3000 µg CO 2 /h) in the mycelium of F. fomentarius s. str., F. inzengae, F. pinicola, and 2-3 times lower (does not exceed 1000 µg CO 2 /h) in the mycelium F. betulina and Ph.igniarius (Tables 1-3).
The total emission activity of the mycelium depends not only on temperature but also on its size.For example, at 20 • C, an increase in the mycelium area of F. betulina by 1.6 times (from 13 to 21 cm 2 ) is accompanied by a similar 1.6-fold increase in TEA (from 303 to 490 µg CO 2 /h).An increase in mycelium size in Ph. igniarius by 1.5 times (from 6 to 9 cm 2 ) at 20 • C leads to a rise in its TEA by 1.6 times (Table 1).In F. pinicola at 20 • C, an increase in the size of the mycelium by 1.2-1.3times is accompanied by an increase in TEA by 1.2-1.5 times (Table 2).An increase in TEA proportional to the increase in mycelium area is also observed in the F. fomentarius s. str.and F. inzengae strains (Table 3).
The size of the mycelium reflects the intensity of its growth, which is positively related to temperature.The correlation coefficient of the daily increase in mycelium area with temperature for F. betulina is 0.61, F. pinicola is 0.57 (strain collected on Picea)-0.76(strain collected on Betula), F. inzengae and Ph.igniarius 0.85, and F. fomentarius s. str.0.97.The temperature coefficient (Q 10 ) of mycelium growth with an increase in temperature from 10 to 20 • C varies from 1.3 (F.inzengae) to 2.0 (F.fomentarius s. str.) and on average is 1.5; it has the same average value when the temperature increases from 20 to 30 • C. At 30 • C, the growth rate of F. betulina and Ph.igniarius mycelium reaches its maximum, as well as F. pinicola, F. inzengae, F. fomentarius s. str. at 35 • C (Tables 1-3).

Discussion
There is an opinion that for the majority of representatives of the boreal microbiota, adapted to an average summer temperature of about +15 • C, an increase in temperature to 30 • C will be tantamount to temperature shock [5].However, as the results of this study show, in the range of 10-30 • C, xylotrophic fungi respond positively to increased temperature.Thus, in this range, the specific CO 2 emission activity of the mycelium, which is an indicator of its respiratory activity, increases on average 1.7 times with an increase in temperature of 10 • C and 3 times with its increase from 10 to 30 • C: 1.7 × 1.7 = 2.9.In other words, the specific CO 2 emission activity of the mycelium of xylotrophic fungi obeys the Van't Hoff rule, and this determines the linear nature of its temperature dynamics.The specific emission activity of the mycelium does not depend on the size of the mycelium, and its relationship with temperature is described by the following Equation ( 5): where SEA T1 and SEA T2 are specific CO 2 emission at temperature T 1 and T 2 , respectively; Q 10SEA -temperature coefficient of specific CO 2 emission activity.Thus, the only driver of the specific CO 2 emission activity of the mycelium of xylotrophic fungi is temperature, or rather its temperature sensitivity, the indicator of which is the temperature coefficient.The latter, in the range of 10-30 • C, varies depending on the species from 1.2 to 2.1, with an average of 1.7.We also recorded similar Q 10 values of specific CO 2 emission activity when analyzing the gas exchange of wood residues destroyed by xylotrophic fungi: 2.0-2.1 [11].The close positive relationship between specific emission activity and temperature determines its unstable nature.One of the results of this is a daily fluctuation in the intensity of CO 2 gas exchange of wood residues: an increase in the daytime and a decrease in the night.
The total CO 2 emission activity of the mycelium is determined by its specific emission activity and size.Accordingly, its temperature dynamics have two drivers: the temperature sensitivity of (a) specific emission and (b) mycelium growth, indicators of which are the temperature coefficients of specific emission and mycelium growth.The relationship between total emission and temperature is described by the following Equation ( 6): where TEA T2 and TEA T1 are the total CO 2 emission at temperature T 1 and T 2 , respectively; Q 10 SEA and Q 10 SM are the temperature coefficient of specific CO 2 emission and mycelium growth, respectively.Depending on the species of fungi, the Q 10 of mycelium growth ranges from 1.3 to 2.0.On average, it is 1.5 and almost identical to Q 10 of specific emission: 1.7.Therefore, an increase in temperature in the range of 10-30 • C causes an almost equal increase in two unidirectional processes-specific CO 2 emission activity and mycelium growth.Their joint action causes an exponential increase in total CO 2 emissions.The dependence on the mycelium size determines another very important feature of the total CO 2 emission activity of xylotrophic fungi.Because the growth of mycelium means an irreversible increase in its size and mass, the temperature dynamics of the total emission also have the character of a directed, irreversible process.The total emission reaches its maximum at 30-35 • C, a temperature at which both the maximum specific CO 2 emission activity and the size of the mycelium are observed.

Conclusions
In the range of summer temperatures (10-30 • C) that are relevant for temperate latitudes, the CO 2 emission activity of xylotrophic fungi is closely and positively related to temperature.Their specific CO 2 emission activity is determined by the respiratory activity of the mycelium and does not depend on its size.The only driver of specific emissions is temperature, an increase in which causes its proportional (linear) increase.The total CO 2 emission activity, which is an indicator of the amount of CO 2 emitted, depends on the size and specific emission activity of the mycelium.It has the character of an irreversible, directional process that increases exponentially with increasing temperature to 30-35 • C.This gives fairly strong grounds to assume that climate warming will lead to an exponential increase in the CO 2 emission activity of woody debris, which, in turn, could potentially contribute to the acceleration of climate change.

Table 1 .
The total and specific CO2 emission activity of the dikaryotic mycelium of Fomitopsis betulina and Phellinus igniarius and its relationship with their area and temperature.Temperature,

Table 1 .
The total and specific CO 2 emission activity of the dikaryotic mycelium of Fomitopsis betulina and Phellinus igniarius and its relationship with their area and temperature.

Table 2 .
The total and specific CO 2 emission activity of the dikaryotic mycelium of Fomitopsis pinicola collected on Betula and Picea and its relationship with their area and temperature.

Table 3 .
The total and specific CO 2 emission activity of the dikaryotic mycelium of Fomes fomentarius sensu stricto and Fomes inzengae and its relationship with their area and temperature.