Microbial communities and activities of forest floor fractions in degraded pine forests after thinning treatments

Summary Litter decomposition is a key process of biogeochemical cycling in forest ecosystems, which has an impact on biodiversity, nutrient availability, greenhouse gas emissions, soil organic matter and carbon sequestration. The objective ot the study was the evaluation of forest floor composition at three decomposition stage, considering: i) C and N amount; ii) the abundance and composition of microbial communities (bacteria and fungi); iii) the hydrolytic enzyme activities; iv) the CO2 emissions. We hypothesized changes of structural and functional diversity during the decomposition processes, affecting in turn carbon and nitrogen cycling. The study has been conducted in a degraded peri-urban pine forest of central Italy in the frame of the FoResMit LIFe project. Forest floor has been collected under three management regimes: i) traditional thinning, ii) selective thinning and iii) no thinning. Leaf (L), fragmented (F) and humified (H) litter fractions have been separated considering the different decomposition stage. A significant decrease of carbon content and C/N ratio has been observed along decomposition process. This matched with changes in bacterial community composition, as well as with the increase of fungal richness and diversity, and enzyme activity. Forest floor contributed between 18 and 37% of total CO2 efflux from soil, on average. Thinning increased forest floor pool without affecting microbial composition, diversity and activity. Material and methods The study area is the peri-urban forest of Monte Morello (43°51′20′′N; 11°14′23′′E), located in the Sesto Fiorentino municipality and close to the urban area of Florence in Tuscany Region. This forest is the result of the reforestations activities realized from 1909 to 1980; specifically experimental plots are 50 to 60 years old. The main tree species used are Pinus nigra J.F. Arnold, Pinus brutia Ten. subsp. brutia, Cupressus sempervirens, Fraxinus ornus L. , Quercus cerris L. and Quercus pubescens L. After reforestation the stands have been abandoned with consequences on trees stability, mortality, absence of regeneration, marked susceptibility to adversities and increase of fire risk (Cenni et al., 1998). Between September and December 2016, three silvicultural treatments have been tested in replicated plots of 1.5 ha approx. with the objective of restoring the ecological stability and enhance the resistance and resilience of the forest: traditional (negative selection) and selective (positive selection) thinning, plus three control plots without intervention (Paletto et al., 2017). Forest floor has been collected in January 2017, after thinning operations, by pressing a 600-700 cm2 steel sheet sampling frame 10 cm deep (or similar) into the forest floor and collecting all litter material above the soil. After sampling, forest floor has been separated into three components: leaf – L (fresh or slightly discoloured, with no or weak breaking up, material), fragmented – F (medium to strongly fragmented material with many mycelia and thin roots), humified – H (completely decomposed amorphous material). Samples have been dried and homogenized at 0.5 mm with a cut-mill for carbon and nitrogen content determination with an elemental analyzer (Flash 2000, Thermo Fisher). CO2 emissions were measured in two replicates for each of the 9 plots with a portable IRGA (EGM4, PP system) from collars with a without forest floor from April to December 2017. Enzyme activity was measured according to the methods of Marx et al. (2001) and Vepsäläinen et al. (2001), based on the use of fluorogenic methylumbelliferyl (MUF)-substrates. Soil was analysed for N-acetyl-βglucosaminidase, β-glucosidase, butyrate esterase, acid phosphatase, arylsulphatase, β-xylosidase, cellulose and acetate esterase activity. The composition and structure of the bacterial and fungal communities were estimated by PCR-DGGE exploiting the 16S rDNA of bacteria and 18S rDNA of fungi. Bacterial and fungal community diversity was characterized through two indicators: richness (R, number of bands) and Shannon-Weiner index (H). nMDS was used to represent the similarity distance between each DGGE profile in a two-dimensional space. Results and discussion The three components of forest floor showed well distinguished characteristics, with a decrease of C content along the litter decompositions level (Table 1). A correspondent increase of N % determined a decrease of C/N ratio. C is lost first during decomposition process and the concentration of N increases consequently. This pattern occurred independent of treatments and remained stable along time. Differently, both thinning treatments increased the amount of forest floor (significantly for L and F fractions). L F H biomass kg m -2 N % C % C/N ratio biomass kg m -2 N % C % C/N ratio biomass kg m -2 N % C % C/N ratio control 0.32 a 0.77 46 64 1.42 a 0.97 36 48 0.96 1.14 a 26 23 traditional 0.68 b 0.82 45 60 2.19 b 1.00 36 56 1.34 1.24 a 27 22 selective 0.43 b 0.75 46 64 1.93 b 1.12 40 57 1.17 1.39 a 29 21 Table 1. Biomass, C and N percentage and C/N ratio of forest floor Forest floor contribution to CO2 emissions was highly variable (between 2 and 72 %) and did not follow temperature trend. A lower contribution, on average, was observed in thinned plots. Apr May Jun Jul Aug Sep Oct Nov Dec Average control 4 48 37 24 26 39 21 25 63 37 traditional 12 21 23 30 17 19 4 6 20 18 selective 7 19 18 33 25 23 5 12 42 21 Table 2. Percentage contribution of forest floor to CO2 emissions from soil Enzyme activities and functional diversity increased along decomposition stage, whereas non significant differences due to thinning treatments were observed. Among enzymes, arylsulphatase was the most represented in H fraction (80 %). L F H Enzyme activity SimpsonYule index Enzyme activity SimpsonYule index Enzyme activity SimpsonYule index control 50.81 6.63 54.29 7.10 59.81 7.42 traditional 46.11 6.62 57.68 7.06 51.16 7.47 selective 42.72 6.49 50.54 6.95 56.08 7.39 Table 3. Enzyme activity (mean of 8 enzymes) and functional diversity in the forest floor Bacterial communities did not change significantly in abundance and diversity, whereas the different decomposition stage alters significantly their composition. Differently, fungal communities richness and diversity increased significantly along decomposition process, confirming results of arylsulphatase. Thinning treatments did not affect microbial communities composition and diversity. Bacteria Fungi R H R H L F H L F H L F H L F H control 23.7 20.7 20.3 3.13 2.91 2.90 8.3 c 11.3 bc 14.7 abc 2.08 b 2.38 ab 2.64 a traditional 24.3 17.3 27.0 3.14 2.83 3.25 8.3 bc 12.3 abc 18.7 a 2.10 b 2.48 ab 2.87 a selective 17.0 22.7 19.7 2.75 3.09 2.87 11.0 bc 12.7 abc 15.7 ab 2.37 ab 2.47 ab 2.69 a Table 4. Richness (R) and Shannon index (H) calculated on bacterial and fungal DGGE profiles. Overall, decomposition process was characterized by changes in chemical and microbiological composition, driving higher enzyme activities in more decomposed fractions. Thinning reduced microbial respiration, possibly as a consequence of the enrichment of fresh, low decomposed, organic matter (L and F litter fractions), without affecting microbial diversity and composition. Acknowledgments The work was financially supported by the LIFE program, in the context of FoResMit project (LIFE14/CCM/IT/905) “recovery of degraded coniferous Forests for environmental sustainability Restoration and climate change Mitigation”.