Frank Rasche

Belowground carbon allocation by trees drives seasonal patterns of extracellular enzyme activities by altering microbial community composition in a beech forest soil.

Plant seasonal cycles alter carbon (C) and nitrogen (N) availability for soil microbes, which may affect microbial community composition and thus feed back on microbial decomposition of soil organic material and plant N availability. The temporal dynamics of these plant–soil interactions are, however, unclear. Here, we experimentally manipulated the C and N availability in a beech forest through N fertilization or tree girdling and conducted a detailed analysis of the seasonal pattern of microbial community composition and decomposition processes over 2 yr. We found a strong relationship between microbial community composition and enzyme activities over the seasonal course. Phenoloxidase and peroxidase activities were highest during late summer, whereas cellulase and protease peaked in late autumn. Girdling, and thus loss of mycorrhiza, resulted in an increase in soil organic matter-degrading enzymes and a decrease in cellulase and protease activity. Temporal changes in enzyme activities suggest a switch of the main substrate for decomposition between summer (soil organic matter) and autumn (plant litter). Our results indicate that ectomycorrhizal fungi are possibly involved in autumn cellulase and protease activity. Our study shows that, through belowground C allocation, trees significantly alter soil microbial communities, which may affect seasonal patterns of decomposition processes.

Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica

Leaf surfaces (phyllospheres) have been shown to provide appropriate conditions for colonization by microorganisms including diazotrophic bacteria that are able to fix atmospheric nitrogen (N2). In this study, we determined leaf-associated N2 fixation of a range of rainforest plants in Costa Rica, under different environmental conditions, by tracing biomass N incorporation from 15N2. N2-fixing bacterial communities of the plant species Carludovica drudei, Grias cauliflora and Costus laevis were investigated in more detail by analysis of the nifH gene and leaf-associated bacteria were identified by 16S rRNA gene analysis. N2 fixation rates varied among plant species, their growth sites (different microclimatic conditions) and light exposure. Leaf-associated diazotrophic bacterial communities detected on C. drudei and C. laevis were mainly composed of cyanobacteria (Nostoc spp.), whereas on the leaves of G. cauliflora γ-proteobacteria were dominant in addition to cyanobacteria. The complexity of diazotrophic communities on leaves was not correlated with N2 fixation activity. 16S rRNA gene sequence analysis suggested the presence of complex microbial communities in association with leaves, however, cyanobacteria showed only low abundance. Our findings suggest that cyanobacteria as well as γ-proteobacteria associated with leaf-colonizing epiphytes may provide significant nitrogen input into this rainforest ecosystem.

Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant. Two field experiments were carried out in Spain to analyze the potential effects of conventional and genetically modified T4-lysozyme producing potatoes on shoot-associated bacteria. The first baseline field trial 2002 was performed in Meliana in which three conventional potato lines, Achirana Inta, Desirée, and Merkur, were cultivated and sampled at flowering. The second field trial was conducted in Cella in 2003 in order to compare the effects of a senescent transgenic, T4 lysozyme expressing potato trait, Desirée DL 12, with its isogenic, non-transformed parental line Desirée. Structural characteristics of potato shoot-associated bacteria was assayed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and dominant community members within T-RFLP profiles were identified by sequence analysis of generated 16S rRNA gene libraries. Cultivable bacteria isolated from shoots of potatoes grown in the Meliana field trial were monitored for antibiosis against Ralstonia solanacearum, whereas isolates derived from shoots of potatoes cultivated in the Cella trial were screened for antagonism against Ralstonia solanacearum and Rhizoctonia solani, and for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Determined antagonists were identified by 16S rRNA gene analysis. All potato traits hosted a cultivar-specific community of bacteria with antagonism against the pathogens and/or potential to produce ACC deaminase. Several antagonists obtained from the Cella field potatoes were also observed as ACC deaminase producers. Community profiling revealed a greater diversity differentiation between the senescent T4 lysozyme expressing and parental Desirée lines grown in the Cella field as compared to the variations between the three flowering conventional lines cultivated in the Meliana field trial. Effects of the two varying field sites and different vegetation stages were greater than those of T4 lysozyme when investigating the community composition of bacteria colonizing the shoots of the Desirée line cultivated in both field trials.

The molecular microbial perspective of organic matter turnover and nutrient cycling in tropical agroecosystems - What do we know?

A primary goal of low-input small-holder farming systems in the tropics is the appropriate management of organic matter (OM) turnover and nutrient cycling via adapted agricultural practices. These emphasize the promotion of soil organic matter (SOM) turnover and carbon (C) sequestration, nutrient use efficiency and soil microbial activity. Soil microbial communities are acknowledged as key players in the terrestrial C and nutrient (e.g., nitrogen (N) and phosphorus (P)) cycles. They respond sensitively to agricultural management with shifts in their community structure as well as functional properties (i.e., decomposition and mineralization). This may be in particular evident for tropical, agriculturally managed soils which show an accelerated microbial decomposition activity induced by favorable climatic and unique physicochemical soil conditions. Molecular techniques advanced the understanding about the composition of soil microbial communities and partially their functions standing in close interaction with SOM dynamics. So far, such methods have rarely been used for elucidating microbial community dynamics including composition and functioning in tropical soils under agricultural use. The primary objective of this article is thus to summarize the existing literature on tropical soil microbial ecology as drivers of OM turnover and crop nutrient supply in soils under agricultural use. This included the highlighting of the latest efforts in deploying particularly nucleic acid-based, cultivation-independent techniques to study the compositional status of soil microbial decomposer communities and, to a smaller extent, their functional attributes in response to land use change and OM management in tropical agroecosystems. The majority of available studies on tropical microbial ecology so far concentrated primarily on the description of compositional microbial community dynamics. It was, however, hardly questioned if detected structural microbial community changes substantially influenced microbial key processes which actually maintain ecosystem functioning and soil productivity. This merit remains substantially unexplored in tropical soils under agricultural use as altered microbial community compositions may be only transient with time with potentially negligible consequences on relevant microbial functioning. There are, however, a few specialized key functional microbial groups whose presence or absence may actually affect the performance, speed and recovery of important ecosystem processes including the transformation of OM and supply of crop nutrients (e.g., N and P). These may finally regulate and determine the productivity of tropical, low-input small-holder farming systems which rely essentially on indigenous soil fertility. Consequently, research recommendations are discussed with emphasis on unique characteristics of tropical environments and tropical agroecosystems to improve the current understanding about the link between microbial key players and productivity of tropical, agriculturally managed soils.

Biocontrol agent Fusarium oxysporum f.sp. strigae has no adverse effect on indigenous total fungal communities and specific AMF taxa in contrasting maize rhizospheres

We studied the effects of Fusarium oxysporum f.sp. strigae (Fos), a soil-borne biocontrol agent (BCA) against Striga hermonthica, on total fungal and arbuscular mycorrhizal fungal (AMF) taxa in rhizospheres of maize in both clayey and sandy soil. Effects of Fos-BCA ‘Foxy-2’ were evaluated against (1) S. hermonthica presence, and (2) organic fertilization with Tithonia diversifolia residues at 14, 28 and 42 d after ‘Foxy-2’ inoculation, via DNA-based quantitative PCR and TRFLP fingerprinting. In both soils, ‘Foxy-2’ occasionally promoted total fungal abundance, while the community composition was mainly altered by T. diversifolia and S. hermonthica. Notably, ‘Foxy-2’ stimulated AMF Gigaspora margarita abundance, while G. margarita was suppressed by S. hermonthica. Total fungal and AMF abundance were promoted by T. diversifolia residues. In conclusion, ‘Foxy-2’ resulted in no adverse effects on indigenous rhizosphere fungal communities substantiating its environmental safety as BCA against S. hermonthica.

Genetic diversity and symbiotic effectiveness of Bradyrhizobium strains nodulating selected annual grain legumes growing in Ethiopia

Vigna unguiculata, Vigna radiata and Arachis hypogaea growing in Ethiopia are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the genetic identity and symbiotic effectiveness of these bacteria, a collection of 36 test strains originating from the root nodules of the three hosts was investigated using multilocus sequence analyses (MLSA) of core genes including 16S rRNA, recA, glnII, gyrB, atpD and dnaK. Sequence analysis of nodA and nifH genes along with tests for symbiotic effectiveness using δ15N analysis were also carried out. The phylogenetic trees derived from the MLSA grouped most test strains into four well-supported distinct positions designated as genospecies I-IV. The maximum likelihood (ML) tree that was constructed based on the nodA gene sequences separated the entire test strains into two lineages, where the majority of the test strains were clustered on one of a well-supported large branch that comprise Bradyrhizobium species from the tropics. This clearly suggested the monophyletic origin of the nodA genes within the bradyrhizobia of tropical origin. The δ15N-based symbiotic effectiveness test of seven selected strains revealed that strains GN100 (δ15N=0.73) and GN102 (δ15N=0.79) were highly effective nitrogen fixers when inoculated to cowpea, thus can be considered as inoculants in cowpea production. It was concluded that Ethiopian soils are a hotspot for rhizobial diversity. This calls for further research to unravel as yet unknown bradyrhizobia nodulating legume host species growing in the country. In this respect, prospective research should also address the mechanisms of symbiotic specificity that could lead to high nitrogen fixation in target legumes.

Proliferation of the biocontrol agent Fusarium oxysporum f. sp. strigae and its impact on indigenous rhizosphere fungal communities in maize under different agro-ecologies

Our objectives were to (1) monitor the proliferation of the biocontrol agent (BCA) Fusarium oxysporum f. sp. strigae strain “Foxy-2”, an effective soil-borne BCA against the parasitic weed Striga hermonthica, in the rhizosphere of maize under different agro-ecologies, and (2) investigate its impact on indigenous rhizosphere fungal community abundance and composition. Field experiments were conducted in Busia and Homa Bay districts in western Kenya during two cropping seasons to account for effects of soil type, climate, growth stage and seasonality. Maize seeds were coated with or without “Foxy-2” and soils were artificially infested with S. hermonthica seeds. One treatment with nitrogen rich organic residues (Tithonia diversifolia) was established to compensate hypothesized resource competition between “Foxy-2” and the indigenous fungal community. Rhizosphere soil samples collected at three growth stages (i.e., EC30, EC60, EC90) of maize were subjected to abundance measurement of “Foxy-2” and total indigenous fungi using quantitative polymerase chain reaction (qPCR) analysis. Terminal restriction fragment length polymorphism (TRFLP) analysis was used to assess potential alterations in the fungal community composition in response to “Foxy-2” presence. “Foxy-2” proliferated stronger in the soils with a sandy clay texture (Busia) than in those with a loamy sand texture (Homa Bay) and revealed slightly higher abundance in the second season. “Foxy-2” had, however, only a transient suppressive effect on total indigenous fungal abundance which ceased in the second season and was further markedly compensated after addition of T. diversifolia residues. Likewise, community structure of the indigenous fungal community was mainly altered by maize growth stages, but not by “Foxy-2”. In conclusion, no adverse effects of “Foxy-2” inoculation on indigenous fungal rhizosphere communities were observed corroborating the safety of this BCA under the given agro-ecologies.

Soil properties, seasonality and crop growth stage exert a stronger effect on rhizosphere prokaryotes than the fungal biocontrol agent Fusarium oxysporum f.sp. strigae

Highlights • Natural factors had major effects on community dynamics of microbial target groups.• Conversely, “Foxy-2” exposed no major effect on rhizosphere microbial communities.• Archaeal community had greater rhizosphere competence than “Foxy-2” in clayey soil.• Compatibility of indigenous soil nitrifying prokaryotes with “Foxy-2” was verified.

Development of a primer system to study abundance and diversity of the gene coding for alanine aminopeptidase pepN gene in Gram-negative soil bacteria

A new set of primers was developed allowing the specific detection of the pepN gene (coding for alanine aminopeptidase) from Gram-negative bacteria. The primers were designed in silico by sequence alignments based on available DNA sequence data. The PCR assay was validated using DNA from selected pure cultures. The analysis of gene libraries from extracted DNA from different soil samples revealed a high diversity of pepN related sequences mainly related to α-Proteobacteria. Most sequences obtained from clone libraries were closely related to already published sequences (<80% homology on amino acid level), which may be related to the conserved character of the amplified region of pepN. By linking the diversity data obtained by the clone library studies to potential enzymatic activities of alanine aminopeptidase, lowest diversity of pepN was found in those soil samples which displayed lowest activity levels, which confirms the importance of diversity for the ecosystem function mainly when transformation processes of complex molecules are studied.

Biological nitrification inhibition activity in a soil-grown biparental population of the forage grass, Brachiaria humidicola

AimUtilization of biological nitrification inhibition (BNI) strategy can reduce nitrogen losses in agricultural systems. This study is aimed at characterizing BNI activity in a plant-soil system using a biparental hybrid population of Brachiaria humidicola (Bh), a forage grass with high BNI potential but of low nutritional quality.MethodsSoil nitrification rates and BNI potential in root-tissue were analyzed in a hybrid population (117), obtained from two contrasting Bh parents, namely CIAT 26146 and CIAT 16888, with low and high BNI activity, respectively. Observed BNI activity was validated by measuring archaeal (AOA) and bacterial (AOB) nitrifier abundance in the rhizosphere soil of parents and contrasting hybrids. Comparisons of the BNI potential of four forage grasses were conducted to evaluate the feasibility of using nitrification rates to measure BNI activity under field and pot grown conditions.ResultsHigh BNI activity was the phenotype most commonly observed in the hybrid population (72%). BNI activity showed a similar tendency for genotypes grown in pots and in the field. A reduction in AOA abundance was found for contrasting hybrids with low nitrification rates and high BNI potential.ConclusionBh hybrids with high levels of BNI activity were identified. Our results demonstrate that the microcosm incubation and the in vitro bioassay may be used as complementary methods for effectively assessing BNI activity. The full expression of BNI potential of Bh genotypes grown in the soil (i.e. low nitrification rates) requires up to one year to develop, after planting.