Evaluation of denaturing gradient gel electrophoresis (DGGE) used to describe structure of bacterial communities in Istrian cheese

Denaturing gradient gel electrophoresis (DGGE) is a powerful method used to study structure of bacterial communities, without cultivation, based on the diversity of the genes coding for ribosomal RNA. However, the results are strongly dependent on the respective target region of the used primer systems. Therefore, three primer pairs that amplify different variable regions of the 16S rRNA gene (V1, V3 and V6 to V8) were tested in order to investigate the bacterial diversity existent in Istrian cheese. We found that primer set extremely influenced DGGE analysis. V3 primers were most efficient when 15 cheese associated isolates were resolved by DGGE. However, for Istrian cheese analysis, the best separation and highest number of bands in DGGE patterns were noticed for V6 to V8 primer pairs.Key words: Denaturing gradient gel electrophoresis, bacterial communities, Istrian cheese

Dynamics of Bacterial Root Endophytes of Malus domestica Plants Grown in Field Soils Affected by Apple Replant Disease

Apple replant disease (ARD) is a worldwide problem for tree nurseries and orchards leading to reduced plant growth and fruit quality. The etiology of this complex phenomenon is poorly understood, but shifts of the bulk soil and rhizosphere microbiome seem to play an important role. Since roots are colonized by microbes from the rhizosphere, studies of the endophytic microbiome in relation to ARD are meaningful. In this study, culture-independent and culture-dependent approaches were used in order to unravel the endophytic root microbiome of apple plants 3, 7, and 12 months after planting in ARD-affected soil and ARD-unaffected control soil at two different field sites. Next to a high diversity of Pseudomonas in roots from all soils, molecular barcoding approaches revealed an increase in relative abundance of endophytic Actinobacteria over time in plants grown in ARD and control plots. Furthermore, several amplicon sequence variants (ASVs) linked to Streptomyces, which had been shown in a previous greenhouse ARD biotest to be negatively correlated to shoot length and fresh mass, were also detected in roots from both field sites. Especially in roots of apple plants from control soil, these Streptomyces ASVs increased in their relative abundance over time. The isolation of 150 bacterial strains in the culture-dependent approach revealed a high diversity of members of the genus Pseudomonas, confirming the data of the molecular barcoding approach. However, only partial overlaps were found between the two approaches, underlining the importance of combining these methods in order to better understand this complex disease and develop possible countermeasures. Overall, this study suggests a key role of Streptomyces in the etiology of ARD in the field. Copyright © 2022 Mahnkopp-Dirks, Radl, Kublik, Gschwendtner, Schloter and Winkelmann

Effect of Soil Tillage Practices on Dynamic of Bacterial Communities in Soil

Several studies have indicated that intensive tillage has notable effect on properties of the soil microbiota that may influence numerous important soils functions, e.g. mobilization of nutrients or change of the overall emission rates of greenhouse gases. Therefore, the aim of our study was to investigate dynamic of microbial communities in soil planted with soybean under different tillage systems. Moreover, abundance of populations harboring the nitrous- oxide reductase gene (nosZ) as an indicator for potential shift s in N2O emission rates was studied. The study was established at chernozem soil of Northern Baranja region in Republic of Croatia as completely randomized block design of four replicate plots for each tillage system in three years experiment. The soil was managed as followed: CT - conventional tillage (moldboard ploughing at 25-30 cm depth), DH - multiple discs harrowing (10-15 cm depth), and NT – no-tillage system. Soil samples were collected in summer and autumn in year 2003. Our results suggested that the reduction of tillage had no effects on the bacterial community structure. This might be a result of the very dry climatic conditions at the investigated site and /or a result of plant species effect (soybean). Slight effects of the tillage management became visible at least when samples were taken in autumn for microbes harboring the N2O reductase gene, indicating that there might be shift s in denitrification pattern in response to changes in tillage practice

Impact of high carbon amendments and pre-crops on soil bacterial communities

A 2-year outdoor mesocosm experiment was carried out to determine the effects of high C amendments (HCAs; wheat straw and sawdust) compared to a control with no addition of HCAs (no-HCA) and 2 different crop rotation systems (spring barley/winter barley and faba bean/winter barley) on soil bacterial communities using a molecular barcoding approach. Samples were analyzed after pre-crop harvest (T1) and harvest of winter barley (T2). Our data demonstrate a clear drop in bacterial diversity after winter barley harvest in the no-HCA and wheat straw treatment compared to the pre-crops. Sawdust application had a stabilizing effect on bacterial diversity compared to the pre-crops and induced an increase in carbon (C) stocks in soil which were however negatively correlated with yields. Main responders in the no-HCA and wheat straw treatment compared to the pre-crops were bacteria of the phyla Actinobacteria and Bacteroidetes which were enriched and bacteria belonging to Firmicutes, Gemmatimonadetes, Proteobacteria, and Gemmatimonadaceae which were depleted. Overall differences between wheat straw-amended and no-HCA control samples were small and included single ASVs from various phyla. In sawdust-amended samples, only a shift of some Proteobacteria families was observed compared to the no-HCA control. Overall, pre-crop plant species had small influence on the observed response pattern of the soil microbiome towards the amendments and was only visible for wheat straw

Molecular Barcoding Reveals the Genus Streptomyces as Associated Root Endophytes of Apple (Malus domestica) Plants Grown in Soils Affected by Apple Replant Disease

Apple replant disease (ARD) occurs when apple is repeatedly planted at the same site, leading to growth reductions and losses in fruit yield and quality. Up to now, the etiology has been poorly understood; however, soil (micro)biota are known to be involved. Because endophytes often colonize plants via the rhizosphere, this study aimed at comparing the bacterial endophytic root microbiome in plants growing in ARD-affected and unaffected soils from three different sites based on greenhouse biotests using a molecular barcoding approach. The initial endophytic microbiome of the starting material (in vitro propagated plants of the apple rootstock M26) did not significantly affect the overall richness and diversity of the endophytic community in plants after 8 weeks of growth in the respective soils but some genera of the initial microbiome managed to establish in apple roots. Proteobacteria was the dominant phylum in all samples. No differences in diversity or number of amplicon sequence variants (ASVs) between plants grown in ARD soil and unaffected soil was observed. However, several ASVs of high abundance uniquely found in plants grown in ARD-affected soils were Streptomyces spp. In soil from all three sites, these Streptomyces spp. were negatively correlated with plant growth parameters. Future inoculation experiments using selected Streptomyces isolates have to prove whether bacteria from this genus are opportunists or part of the ARD complex. For the first time, the bacterial endophytic community of apple roots grown in ARD-affected soils was characterized, which will help us to understand the etiology of ARD and develop countermeasures

Enrichment of endophytic Actinobacteria in roots and rhizomes of <i>Miscanthus × giganteus</i> plants exposed to diclofenac and sulfamethoxazole

This study investigates how wastewater containing 2 mg l(-1) of sulfamethoxazole (SMX) and 2 mg l(-1) of diclofenac (DCF) affects the composition of bacterial communities present in the roots and rhizomes of Miscanthus x giganteus plants grown in laboratory-scale constructed wetlands. Bacterial communities in plant roots and rhizomes were identified in treated and control samples by 16S rRNA amplicon sequencing. Moreover, bacterial endophytes were isolated in R2A and 1/10 869 media and screened for their ability to metabolize SMX and DCF in liquid medium by HPLC. Our results show significant changes in the abundance of main genera, namely Sphingobium and Streptomyces between control and treated plants. Around 70% of the strains isolated from exposed plants belonged to the phylum Actinobacteria and were classified as Streptomyces, Microbacterium, and Glycomyces. In non-exposed plants, Proteobacteria represented 43.5% to 63.6% of the total. We identified 17 strains able to remove SMX and DCF in vitro. From those, 76% were isolated from exposed plants. Classified mainly as Streptomyces, they showed the highest SMX (33%) and DCF (41%) removal efficiency. These isolates, alone or in combination, might be used as bio-inoculants in constructed wetlands to enhance the phytoremediation of SMX and DCF during wastewater treatment

Eff ect of Soil Tillage Practices on Dynamic of Bacterial Communities in Soil

Summary Several studies have indicated that intensive tillage has notable eff ect on properties of the soil microbiota that may infl uence numerous important soils functions, e.g. mobilization of nutrients or change of the overall emission rates of greenhouse gases. Th erefore, the aim of our study was to investigate dynamic of microbial communities in soil planted with soybean under diff erent tillage systems. Moreover, abundance of populations harboring the nitrous-oxide reductase gene (nosZ) as an indicator for potential shift s in N 2 O emission rates was studied. Th e study was established at chernozem soil of Northern Baranja region in Republic of Croatia as completely randomized block design of four replicate plots for each tillage system in three years experiment. Th e soil was managed as followed: CT -conventional tillage (moldboard ploughing at 25-30 cm depth), DH -multiple discs harrowing (10-15 cm depth), and NT -no-tillage system. Soil samples were collected in summer and autumn in year 2003. Our results suggested that the reduction of tillage had no eff ects on the bacterial community structure. Th is might be a result of the very dry climatic conditions at the investigated site and /or a result of plant species eff ect (soybean). Slight eff ects of the tillage management became visible at least when samples were taken in autumn for microbes harboring the N 2 O reductase gene, indicating that there might be shift s in denitrifi cation pattern in response to changes in tillage practice

Cultivar-specific dynamics: unravelling rhizosphere microbiome responses to water deficit stress in potato cultivars

Abstract Background Growing evidence suggests that soil microbes can improve plant fitness under drought. However, in potato, the world’s most important non-cereal crop, the role of the rhizosphere microbiome under drought has been poorly studied. Using a cultivation independent metabarcoding approach, we examined the rhizosphere microbiome of two potato cultivars with different drought tolerance as a function of water regime (continuous versus reduced watering) and manipulation of soil microbial diversity (i.e., natural (NSM), vs. disturbed (DSM) soil microbiome). Results Water regime and soil pre-treatment showed a significant interaction with bacterial community composition of the sensitive (HERBST) but not the resistant cultivar (MONI). Overall, MONI had a moderate response to the treatments and its rhizosphere selected Rhizobiales under reduced watering in NSM soil, whereas Bradyrhizobium, Ammoniphilus, Symbiobacterium and unclassified Hydrogenedensaceae in DSM soil. In contrast, HERBST response to the treatments was more pronounced. Notably, in NSM soil treated with reduced watering, the root endophytic fungus Falciphora and many Actinobacteriota members (Streptomyces, Glycomyces, Marmoricola, Aeromicrobium, Mycobacterium and others) were largely represented. However, DSM soil treatment resulted in no fungal taxa and fewer enrichment of these Actinobacteriota under reduced watering. Moreover, the number of bacterial core amplicon sequence variants (core ASVs) was more consistent in MONI regardless of soil pre-treatment and water regimes as opposed to HERBST, in which a marked reduction of core ASVs was observed in DSM soil. Conclusions Besides the influence of soil conditions, our results indicate a strong cultivar-dependent relationship between the rhizosphere microbiome of potato cultivars and their capacity to respond to perturbations such as reduced soil moisture. Our study highlights the importance of integrating soil conditions and plant genetic variability as key factors in future breeding programs aiming to develop drought resistance in a major food crop like potato. Elucidating the molecular mechanisms how plants recruit microbes from soil which help to mitigate plant stress and to identify key microbial taxa, which harbour the respective traits might therefore be an important topic for future research