Impact of Cellulose-Rich Organic Soil Amendments on Growth Dynamics and Pathogenicity of Rhizoctonia solani.

Cellulose-rich amendments stimulate saprotrophic fungi in arable soils. This may increase competitive and antagonistic interactions with root-infecting pathogenic fungi, resulting in lower disease incidence. However, cellulose-rich amendments may also stimulate pathogenic fungi with saprotrophic abilities, thereby increasing plant disease severity. The current study explores these scenarios, with a focus on the pathogenic fungus Rhizoctonia solani. Saprotrophic growth of R. solani on cellulose-rich materials was tested in vitro. This confirmed paper pulp as a highly suitable substrate for R. solani, whereas its performance on wood sawdusts varied with tree species. In two pot experiments, the effects of amendment of R. solani-infected soil with cellulose-rich materials on performance of beetroot seedlings were tested. All deciduous sawdusts and paper pulp stimulated soil fungal biomass, but only oak, elder and beech sawdusts reduced damping-off of beetroot. Oak sawdust amendment gave a consistent stimulation of saprotrophic Sordariomycetes fungi and of seedling performance, independently of the time between amendment and sowing. In contrast, paper pulp caused a short-term increase in R. solani abundance, coinciding with increased disease severity for beet seedlings sown immediately after amendment. However, damping-off of beetroot was reduced if plants were sown two or four weeks after paper pulp amendment. Cellulolytic bacteria, including Cytophagaceae, responded to paper pulp during the first two weeks and may have counteracted further spread of R. solani. The results showed that fungus-stimulating, cellulose-rich amendments have potential to be used for suppression of R. solani. However, such amendments require a careful consideration of material choice and application strategy.

Evaluation of Phenolic Root Exudates as Stimulants of Saptrophic Fungi in the Rhizosphere.

The rhizosphere microbial community of crop plants in intensively managed arable soils is strongly dominated by bacteria, especially in the initial stages of plant development. In order to establish more diverse and balanced rhizosphere microbiomes, as seen for wild plants, crop variety selection could be based on their ability to promote growth of saprotrophic fungi in the rhizosphere. We hypothesized that this can be achieved by increasing the exudation of phenolic acids, as generally higher fungal abundance is observed in environments with phenolic-rich inputs, such as exudates of older plants and litter leachates. To test this, a rhizosphere simulation microcosm was designed to establish gradual diffusion of root exudate metabolites from sterile sand into arable soil. With this system, we tested the fungus-stimulating effect of eight phenolic acids alone or in combination with primary root metabolites. Ergosterol-based fungal biomass measurements revealed that most phenolic acids did not increase fungal abundance in the arable soil layer. These results were supported by comparison of fungal biomass in the rhizosphere of wild type Arabidopsis thaliana plants and mutants with altered phenolic acid metabolism. Salicylic acid was the only phenolic acid that stimulated a higher fungal biomass in the arable soil layer of microcosms, but only when combined with a background of primary root metabolites. However, such effect on rhizosphere fungi was not confirmed for a salicylic acid-impaired A. thaliana mutant. For three phenolic acid treatments (chlorogenic acid, salicylic acid, vanillic acid) fungal and bacterial community compositions were analyzed using amplicon sequencing. Despite having little effect on fungal biomass, phenolic acids combined with primary metabolites promoted a higher relative abundance of soil-borne fungi with the ability to invade plant roots (Fusarium, Trichoderma and Fusicolla spp.) in the simulated rhizosphere. Bacterial community composition was also affected by these phenolic acids. Although this study indicates that phenolic acids do not increase fungal biomass in the rhizosphere, we highlight a potential role of phenolic acids as attractants for root-colonizing fungi.

Stimulated saprotrophic fungi in arable soil extend their activity to the rhizosphere and root microbiomes of crop seedlings.

Saprotrophic fungi play an important role in ecosystem functioning and plant performance, but their abundance in intensively managed arable soils is low. Saprotrophic fungal biomass in arable soils can be enhanced with amendments of cellulose-rich materials. Here, we examined if sawdust-stimulated saprotrophic fungi extend their activity to the rhizosphere of crop seedlings and influence the composition and activity of other rhizosphere and root inhabitants. After growing carrot seedlings in sawdust-amended arable soil, we determined fungal and bacterial biomass and community structure in roots, rhizosphere and soil. Utilization of root exudates was assessed by stable isotope probing (SIP) following 13 CO2 -pulse-labelling of seedlings. This was combined with analysis of lipid fatty acids (PLFA/NLFA-SIP) and nucleic acids (DNA-SIP). Sawdust-stimulated Sordariomycetes colonized the seedling's rhizosphere and roots and actively consumed root exudates. This did not reduce the abundance and activity of bacteria, yet higher proportions of α-Proteobacteria and Bacteroidia were seen. Biomass and activity of mycorrhizal fungi increased with sawdust amendments, whereas exudate consumption and root colonization by functional groups containing plant pathogens did not change. Sawdust amendment of arable soil enhanced abundance and exudate-consuming activity of saprotrophic fungi in the rhizosphere of crop seedlings and promoted potential beneficial microbial groups in root-associated microbiomes.

Volatile-mediated antagonism of soil bacterial communities against fungi.

Competition is a major type of interaction between fungi and bacteria in soil and is also an important factor in suppression of plant diseases caused by soil-borne fungal pathogens. There is increasing attention for the possible role of volatiles in competitive interactions between bacteria and fungi. However, knowledge on the actual role of bacterial volatiles in interactions with fungi within soil microbial communities is lacking. Here, we examined colonization of sterile agricultural soils by fungi and bacteria from non-sterile soil inoculums during exposure to volatiles emitted by soil-derived bacterial communities. We found that colonization of soil by fungi was negatively affected by exposure to volatiles emitted by bacterial communities whereas that of bacteria was barely changed. Furthermore, there were strong effects of bacterial community volatiles on the assembly of fungal soil colonizers. Identification of volatile composition produced by bacterial communities revealed several compounds with known fungistatic activity. Our results are the first to reveal a collective volatile-mediated antagonism of soil bacteria against fungi. Given the better exploration abilities of filamentous fungi in unsaturated soils, this may be an important strategy for bacteria to defend occupied nutrient patches against invading fungi. Another implication of our research is that bacterial volatiles in soil atmospheres can have a major contribution to soil fungistasis.

Antifungal Rhizosphere Bacteria Can increase as Response to the Presence of Saprotrophic Fungi.

Knowledge on the factors that determine the composition of bacterial communities in the vicinity of roots (rhizosphere) is essential to understand plant-soil interactions. Plant species identity, plant growth stage and soil properties have been indicated as major determinants of rhizosphere bacterial community composition. Here we show that the presence of saprotrophic fungi can be an additional factor steering rhizosphere bacterial community composition and functioning. We studied the impact of presence of two common fungal rhizosphere inhabitants (Mucor hiemalis and Trichoderma harzianum) on the composition of cultivable bacterial communities developing in the rhizosphere of Carex arenaria (sand sedge) in sand microcosms. Identification and phenotypic characterization of bacterial isolates revealed clear shifts in the rhizosphere bacterial community composition by the presence of two fungal strains (M. hiemalis BHB1 and T. harzianum PvdG2), whereas another M. hiemalis strain did not show this effect. Presence of both M. hiemalis BHB1 and T. harzianum PvdG2 resulted in a significant increase of chitinolytic and (in vitro) antifungal bacteria. The latter was most pronounced for M. hiemalis BHB1, an isolate from Carex roots, which stimulated the development of the bacterial genera Achromobacter and Stenotrophomonas. In vitro tests showed that these genera were strongly antagonistic against M. hiemalis but also against the plant-pathogenic fungus Rhizoctonia solani. The most likely explanation for fungal-induced shifts in the composition of rhizosphere bacteria is that bacteria are being selected which are successful in competing with fungi for root exudates. Based on the results we propose that measures increasing saprotrophic fungi in agricultural soils should be explored as an alternative approach to enhance natural biocontrol against soil-borne plant-pathogenic fungi, namely by stimulating indigenous antifungal rhizosphere bacteria.

Legacy effects of anaerobic soil disinfestation on soil bacterial community composition and production of pathogen-suppressing volatiles.

There is increasing evidence that microbial volatiles (VOCs) play an important role in natural suppression of soil-borne diseases, but little is known on the factors that influence production of suppressing VOCs. In the current study we examined whether a stress-induced change in soil microbial community composition would affect the production by soils of VOCs suppressing the plant-pathogenic oomycete Pythium. Using pyrosequencing of 16S ribosomal gene fragments we compared the composition of bacterial communities in sandy soils that had been exposed to anaerobic disinfestation (AD), a treatment used to kill harmful soil organisms, with the composition in untreated soils. Three months after the AD treatment had been finished, there was still a clear legacy effect of the former anaerobic stress on bacterial community composition with a strong increase in relative abundance of the phylum Bacteroidetes and a significant decrease of the phyla Acidobacteria, Planctomycetes, Nitrospirae, Chloroflexi, and Chlorobi. This change in bacterial community composition coincided with loss of production of Pythium suppressing soil volatiles (VOCs) and of suppression of Pythium impacts on Hyacinth root development. One year later, the composition of the bacterial community in the AD soils was reflecting that of the untreated soils. In addition, both production of Pythium-suppressing VOCs and suppression of Pythium in Hyacinth bioassays had returned to the levels of the untreated soil. GC/MS analysis identified several VOCs, among which compounds known to be antifungal, that were produced in the untreated soils but not in the AD soils. These compounds were again produced 15 months after the AD treatment. Our data indicate that soils exposed to a drastic stress can temporarily lose pathogen suppressive characteristics and that both loss and return of these suppressive characteristics coincides with shifts in the soil bacterial community composition. Our data are supporting the suggested importance of microbial VOCs in the natural buffer of soils against diseases caused by soil-borne pathogens.

Methods for Baiting and Enriching Fungus-Feeding (Mycophagous) Rhizosphere Bacteria.

Mycophagous soil bacteria are able to obtain nutrients from living fungal hyphae. However, with exception of the soil bacterial genus Collimonas, occurrence of this feeding strategy has not been well examined. Evaluation of the importance of mycophagy in soil bacterial communities requires targeted isolation methods. In this study, we compared two different approaches to obtain mycophagous bacteria from rhizospheric soil. A short-term method based on baiting for bacteria that can rapidly adhere to fungal hyphae and a long-term method based on the enrichment of bacteria on fungal hyphae via repeated transfer. Hyphae-adhering bacteria were isolated, identified by 16S rDNA sequencing and tested for antifungal activity and the ability to feed on fungi as the sole source of carbon. Both methods yielded a range of potentially mycophagous bacterial isolates with little phylogenetic overlap. We also found indications for feeding preferences among the potentially mycophagous bacteria. Our results indicate that mycophagy could be an important growth strategy for rhizosphere bacteria. To our surprise, we found several potential plant pathogenic bacteria among the mycophagous isolates. We discuss the possible benefits that these bacteria might gain from colonizing fungal hyphae.

Impact of Collimonas bacteria on community composition of soil fungi.

The genus Collimonas consists of soil bacteria that have the potential to grow at the expense of living fungal hyphae. However, the consequences of this mycophagous ability for soil fungi are unknown. Here we report on the development of fungal communities after introduction of collimonads in a soil that had a low abundance of indigenous collimonads. Development of fungal communities was stimulated by addition of cellulose or by introducing plants (Plantago lanceolata). Community composition of total fungi in soil and rhizosphere and of arbuscular mycorrhizal fungi in roots was examined by PCR-DGGE. The introduction of collimonads altered the composition of all fungal communities studied but had no effects on fungal biomass increase, cellulose degrading activity or plant performance. The most likely explanation for these results is that differences in sensitivity of fungal species to the presence of collimonads result in competitive replacement of species. The lab and greenhouse experiments were complemented with a field experiment. Mesh bags containing sterile sand with or without collimonads were buried in an ex-arable field and a forest. The presence of collimonads had an effect on the composition of fungi invading these bags in the ex-arable site but not in the forest site.