Witoon Purahong

Effects of fungal endophytes on grass and non-grass litter decomposition rates

The influence of clavicipitaceous fungal endophytes on grass decomposition rates has been studied through field and laboratory experiments. However, the effects of endophytes on decomposition rates of non-grass species are unclear. This paper reviews research data related to the effects of fungal endophytes on decomposition rates of three litter types: grass, non-grass leaf litter (including spruce needle litter) and non-grass twigs and wood. We discuss how fungal endophytes are involved in, or regulate decomposition rates and may change lifestyles from fungal endophytes to saprotrophs. Classical morphology and molecular approaches together with digestion enzyme studies provide evidence to suggest that some endophyte species switch their ecological roles and adopt a new life style as saprotrophs. We also explore the main mechanisms that explain how fungal endophytes may decelerate decomposition rates and whether it is directly driven by alkaloids. Further research on the role of fungal endophytes in decomposition rates of both grass and non-grass litter is needed, especially those addressing the direct and indirect mechanisms by which endophytes affect decomposition rates.

Life in leaf litter: novel insights into community dynamics of bacteria and fungi during litter decomposition.

Microorganisms play a crucial role in the biological decomposition of plant litter in terrestrial ecosystems. Due to the permanently changing litter quality during decomposition, studies of both fungi and bacteria at a fine taxonomic resolution are required during the whole process. Here we investigated microbial community succession in decomposing leaf litter of temperate beech forest using pyrotag sequencing of the bacterial 16S and the fungal internal transcribed spacer (ITS) rRNA genes. Our results reveal that both communities underwent rapid changes. Proteobacteria, Actinobacteria and Bacteroidetes dominated over the entire study period, but their taxonomic composition and abundances changed markedly among sampling dates. The fungal community also changed dynamically as decomposition progressed, with ascomycete fungi being increasingly replaced by basidiomycetes. We found a consistent and highly significant correlation between bacterial richness and fungal richness (R = 0.76, P < 0.001) and community structure (RMantel = 0.85, P < 0.001), providing evidence of coupled dynamics in the fungal and bacterial communities. A network analysis highlighted nonrandom co‐occurrences among bacterial and fungal taxa as well as a shift in the cross‐kingdom co‐occurrence pattern of their communities from the early to the later stages of decomposition. During this process, macronutrients, micronutrients, C:N ratio and pH were significantly correlated with the fungal and bacterial communities, while bacterial richness positively correlated with three hydrolytic enzymes important for C, N and P acquisition. Overall, we provide evidence that the complex litter decay is the result of a dynamic cross‐kingdom functional succession.

Linking molecular deadwood-inhabiting fungal diversity and community dynamics to ecosystem functions and processes in Central European forests

Fungi play vital roles in the decomposition of deadwood due to their secretion of various enzymes that break down plant cell-wall complexes. The compositions of wood-inhabiting fungal (WIF) communities change over the course of the decomposition process as the remaining mass of wood decreases and both abiotic and biotic conditions of the wood significantly change. It is currently not resolved which substrate-related factors govern these changes in WIF communities and whether such changes influence the deadwood decomposition rate. Here we report a study on fungal richness and community structure in deadwood of Norway spruce and European beech in temperate forest ecosystems using 454 pyrosequencing. Our aims were to disentangle the factors that correspond to WIF community composition and to investigate the links between fungal richness, taxonomically-resolved fungal identity, and microbial-mediated ecosystem functions and processes by analyzing physico-chemical wood properties, lignin-modifying enzyme activities and wood decomposition rates. Unlike fungal richness, we found significant differences in community structure between deadwood of different tree species. The composition of WIF communities was related to the physico-chemical properties of the deadwood substrates. Decomposition rates and the activities of lignin-modifying enzymes were controlled by the succession of the fungal communities and competition scenarios rather than fungal OTU richness. Our results provide further insights into links between fungal community structure and microbial-mediated ecosystem functions and processes.

Changes within a single land-use category alter microbial diversity and community structure: Molecular evidence from wood-inhabiting fungi in forest ecosystems

The impact of changes within a single land-use category or land-use intensity on microbial communities is poorly understood, especially with respect to fungi. Here we assessed how forest management regimes and a change in forest type affect the richness and community structure of wood-inhabiting fungi across Germany. We used molecular methods based on the length polymorphism of the internal transcribed spacers and the 5.8S rRNA gene to assess fungal operational taxonomic units (OTUs). A cloning/sequencing approach was used to identify taxonomic affinities of the fungal OTUs. Overall, 20-24% and 25-27% of native fungal OTUs from forest reserves and semi-natural forests became undetectable or were lost in managed and converted forests, respectively. Fungal richness was significantly reduced during a regeneration phase in age-class beech forests with a high level of wood extraction (P = 0.017), whereas fungal community structures were not significantly affected. Conversion of forests from native, deciduous to coniferous species caused significant changes in the fungal community structure (R = 0.64-0.66, P = 0.0001) and could reduce fungal richness (P < 0.05) which may depend on which coniferous species was introduced. Our results showed that Ascocoryne cylichnium, Armillaria sp., Exophiala moniliae, Hyphodontia subalutacea and Fomes fomentarius, all known for wood-decaying abilities were strongly reduced in their abundances when forests were converted from beech to coniferous. We conclude that changes within a single land-use category can be regarded as a major threat to fungal diversity in temperate forest ecosystems.

Influence of different forest system management practices on leaf litter decomposition rates, nutrient dynamics and the activity of ligninolytic enzymes: a case study from central European forests.

Leaf litter decomposition is the key ecological process that determines the sustainability of managed forest ecosystems, however very few studies hitherto have investigated this process with respect to silvicultural management practices. The aims of the present study were to investigate the effects of forest management practices on leaf litter decomposition rates, nutrient dynamics (C, N, Mg, K, Ca, P) and the activity of ligninolytic enzymes. We approached these questions using a 473 day long litterbag experiment. We found that age-class beech and spruce forests (high forest management intensity) had significantly higher decomposition rates and nutrient release (most nutrients) than unmanaged deciduous forest reserves (P<0.05). The site with near-to-nature forest management (low forest management intensity) exhibited no significant differences in litter decomposition rate, C release, lignin decomposition, and C/N, lignin/N and ligninolytic enzyme patterns compared to the unmanaged deciduous forest reserves, but most nutrient dynamics examined in this study were significantly faster under such near-to-nature forest management practices. Analyzing the activities of ligninolytic enzymes provided evidence that different forest system management practices affect litter decomposition by changing microbial enzyme activities, at least over the investigated time frame of 473 days (laccase, P<0.0001; manganese peroxidase (MnP), P = 0.0260). Our results also indicate that lignin decomposition is the rate limiting step in leaf litter decomposition and that MnP is one of the key oxidative enzymes of litter degradation. We demonstrate here that forest system management practices can significantly affect important ecological processes and services such as decomposition and nutrient cycling.

Uncoupling of microbial community structure and function in decomposing litter across beech forest ecosystems in Central Europe

The widespread paradigm in ecology that community structure determines function has recently been challenged by the high complexity of microbial communities. Here, we investigate the patterns of and connections between microbial community structure and microbially-mediated ecological function across different forest management practices and temporal changes in leaf litter across beech forest ecosystems in Central Europe. Our results clearly indicate distinct pattern of microbial community structure in response to forest management and time. However, those patterns were not reflected when potential enzymatic activities of microbes were measured. We postulate that in our forest ecosystems, a disconnect between microbial community structure and function may be present due to differences between the drivers of microbial growth and those of microbial function.

Comparing fungal richness and community composition in coarse woody debris in Central European beech forests under three types of management

Managing forests by selection cutting is a promising silvicultural technique for maintaining forest biodiversity. Despite the importance of fungi in decomposition and nutrient cycling in forest ecosystems, no study to date has investigated the effects of selection cutting on fungal communities, especially using a culture-independent molecular technique to assess more than just the species that are fruiting at the time of sampling. Based on operational taxonomic units (OTUs) found in coarse woody debris, we compared the richness and community composition of wood-inhabiting fungi from selection cutting, age-class, and unmanaged European beech-dominated forests. We found that fungal OTU richness in selection cutting and unmanaged forests was not significantly different (P > 0.05), but it was higher, in both cases, than that in the age-class forest (P = 0.0002). Fungal community composition was not significantly different among the three forest types (P > 0.05). Abundances of common, wood-inhabiting fungal OTUs in different forest types were significantly correlated: the highest and lowest correlations were found between unmanaged forests and selection cutting (ρ = 0.52, P < 0.0001, n = 94), and between unmanaged and age-class forests (ρ = 0.30, P = 0.0080, n = 79), respectively.

Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.

Validation of a modified Petri-dish test to quantify aggressiveness of Fusarium graminearum in durum wheat

Fusarium graminearum is a common agent causing Fusarium head blight (FHB) on wheat throughout the world. Aggressiveness is crucial for understanding the interaction between host-pathogen in the FHB-wheat system. In this paper, we modified and validated the Petri-dish test originally described by Mesterhazy (Phytopathologische Zeitschrift 93:12–25, 1978) to quantify the aggressiveness of 25 F. graminearum strains using four durum wheat cultivars with different resistance levels for FHB. The results were highly significant and correlated with those obtained using adult plants in the growth chamber and in the field (r = 0.94, P < 0.001 and r = 0.65, P < 0.001, respectively). The Petri-dish test was further investigated for its repeatability and stability in different durum wheat cultivars and highly significant correlation coefficients were obtained (r = 0.90–0.91 (P < 0.001), 0.89–0.95 (P < 0.001), respectively). In this study, we also demonstrated that germination rate reduction and coleoptile length reduction are parameters involved with aggressiveness of F. graminearum. The mean of three disease parameters from the modified Petri-dish method is introduced in this paper as a new parameter for aggressiveness and named “Petri-dish aggressiveness index”. The results obtained reveal that this modified Petri-dish test is rapid, reliable and stable with different durum wheat cultivars, and yields highly significant correlation coefficients with floret and ear inoculations, thus it is suitable to be used for quantification of aggressiveness of F. graminearum.

A better understanding of functional roles of fungi in the decomposition process: using precursor rRNA containing ITS regions as a marker for the active fungal community

Decomposition and biogeochemical cycling are important ecosystem services provided by fungi (Kulhankova et al. 2006; Gange et al. 2007; Trap et al. 2011). Hitherto, different approaches have been used to examine the fungal community and its functional role in the decomposition process. Culture-dependent approaches have limitations because most fungi in environmental samples are likely to be uncultivable or difficult to isolate and such approaches are biased towards fast-growing fungi and affected by media selection (Anderson and Cairney 2004). On the other hand, cultureindependent molecular approaches are used routinely for fungal diversity and community assessment. The main targets of these approaches are the rRNA genes and spacer regions of the RNA operon (rDNA), thus basing diversity and community composition on DNA content. However, some studies show that DNA may have a long life span in the environment (Nielsen et al. 2004; Levy-Booth et al. 2007; Corinaldesi et al. 2008; Pietramellara et al. 2009). DNA can stem from fungal resting structures and spores or even senescent mycelium, all of which would be metabolically inactive at a given sampling time and even permanently. Consequently, fungal community details from DNA carry some memories about the previous community composition, and may poorly reflect the fungi that are active in decomposition processes at the time of sample collection. Additionally, it is possible that dispersed propagules leave their DNA signatures when they are carried there by wind or animals (Stenlid and Gustafsson 2001). The operational taxonomic unit (OTU) richness of decomposer fungal communities revealed by DNA-based community assessment is usually higher than that based on RNA because of the inclusion of dead or inactive fungal DNA (Anderson and Parkin 2007; Rajala et al. 2011). However, there were few cases reported vice versa or no significant differences between OTU richness of the fungal decomposers derived either by DNAor RNA-based approaches (Bastias et al. 2007; Baldrian et al. 2012). These are because some fungal OTUs are only detected using RNA and they compensate the inactive fungi detected by DNA. Nevertheless, we suggest caution that the presence of background communities of dormant or senescent fungi in environmental samples as an artifact is a potentially major hindrance to unbiased interpretation of real functional roles and correlations with enzyme production and environmental parameters. A DNAbased study (Kulhankova et al. 2006; Kubartova et al. 2009) has revealed that a large proportion of ascomycetes produce few oxidative enzymes and so are restricted in their ability to digest complex substances in the late stages of decomposition. Inactive or dead Ascomycota may be remnants of earlier stages of decomposition (Purahong and Hyde 2011; Rajala et al. 2011), which strongly supports the suggestion that there is an inert background fungal community, and that Handling Editor: Ana Rincon