Rhizoctonia solani disease suppression: addition of keratin-rich soil amendment leads to functional shifts in soil microbial communities.

Promoting soil suppressiveness against soil borne pathogens could be a promising strategy to manage crop diseases. One way to increase the suppression potential in agricultural soils is via the addition of organic amendments. This microbe-mediated phenomenon, although not fully understood, prompted our study to explore the microbial taxa and functional properties associated with Rhizoctonia solani disease suppression in sugar beet seedlings after amending soil with a keratin-rich waste stream. Soil samples were analyzed using shotgun metagenomics sequencing. Results showed that both amended soils were enriched in bacterial families found in disease suppressive soils before, indicating that the amendment of keratin-rich material can support the transformation into a suppressive soil. On a functional level, genes encoding keratinolytic enzymes were found to be abundant in the keratin-amended samples. Proteins enriched in amended soils were those potentially involved in the production of secondary metabolites/antibiotics, motility, keratin-degradation, and contractile secretion system proteins. We hypothesize these taxa contribute to the amendment-induced suppression effect due to their genomic potential to produce antibiotics, secrete effectors via the contractile secretion system, and degrade oxalate-a potential virulence factor of R. solani-while simultaneously possessing the ability to metabolize keratin.

Soil Suppressiveness Against Pythium ultimum and Rhizoctonia solani in Two Land Management Systems and Eleven Soil Health Treatments.

The soil microbiome is known to be crucial for the control of soil-borne plant diseases. However, there is still little knowledge on how to modify the soil microbiome to induce or increase disease suppressiveness. In the present study, we applied eleven soil health treatments combined with conventional and organic agricultural management in a long-term field experiment. Suppressiveness against Pythium ultimum and Rhizoctonia solani was assessed in bioassays for 2 years. In addition, the microbiome community composition and microbial abundance were determined. We found that while several treatments changed the microbial community composition compared to the control, only a combination treatment of anaerobic soil disinfestation, hair meal, and compost addition resulted in suppressiveness against P. ultimum. Pythium suppressiveness is likely to have been caused by an increased microbial abundance and activity. Moreover, the increased abundance of several bacterial taxa, such as Pseudomonas sp., Chryseobacterium sp., members of the family Chitinophagaceae, and the fungal genus Mortierella sp. and family Trichosporonaceae, was measured. There was no overall difference in suppressiveness between conventional and organic land management. Also, no suppressiveness against R. solani could be detected. Our results indicate that a treatment combining the reduction of microorganisms followed by a recovery phase with high amounts of organic amendments may be more effective in inducing suppressiveness than treatments consisting of only one of these measures.

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.

Chitin- and Keratin-Rich Soil Amendments Suppress Rhizoctonia solani Disease via Changes to the Soil Microbial Community.

Enhancing soil suppressiveness against plant pathogens or pests is a promising alternative strategy to chemical pesticides. Organic amendments have been shown to reduce crop diseases and pests, with chitin products the most efficient against fungal pathogens. To study which characteristics of organic products are correlated with disease suppression, an experiment was designed in which 10 types of organic amendments with different physicochemical properties were tested against the soilborne pathogen Rhizoctonia solani in sugar beet seedlings. Organic amendments rich in keratin or chitin reduced Rhizoctonia solani disease symptoms in sugar beet plants. The bacterial and fungal microbial communities in amended soils were distinct from the microbial communities in nonamended soil, as well as those in soils that received other nonsuppressive treatments. The Rhizoctonia-suppressive amended soils were rich in saprophytic bacteria and fungi that are known for their keratinolytic and chitinolytic properties (i.e., Oxalobacteraceae and Mortierellaceae). The microbial community in keratin- and chitin-amended soils was associated with higher zinc, copper, and selenium, respectively.IMPORTANCE Our results highlight the importance of soil microorganisms in plant disease suppression and the possibility to steer soil microbial community composition by applying organic amendments to the soil.

Verticillium Wilt in Oilseed Rape-the Microbiome is Crucial for Disease Outbreaks as Well as for Efficient Suppression.

Microbiome management is a promising way to suppress verticillium wilt, a severe disease in Brassica caused by Verticillium longisporum. In order to improve current biocontrol strategies, we compared bacterial Verticillium antagonists in different assays using a hierarchical selection and evaluation scheme, and we integrated outcomes of our previous studies. The result was strongly dependent on the assessment method chosen (in vitro, in vivo, in situ), on the growth conditions of the plants and their genotype. The most promising biocontrol candidate identified was a Brassica endophyte Serratia plymuthica F20. Positive results were confirmed in field trials and by microscopically visualizing the three-way interaction. Applying antagonists in seed treatment contributes to an exceptionally low ecological footprint, supporting efficient economic and ecological solutions to controlling verticillium wilt. Indigenous microbiome, especially soil and seed microbiome, has been identified as key to understanding disease outbreaks and suppression. We suggest that verticillium wilt is a microbiome-driven disease caused by a reduction in microbial diversity within seeds and in the soil surrounding them. We strongly recommend integrating microbiome data in the development of new biocontrol and breeding strategies and combining both strategies with the aim of designing healthy microbiomes, thus making plants more resilient toward soil-borne pathogens.

The structure of the Brassica napus seed microbiome is cultivar-dependent and affects the interactions of symbionts and pathogens.

BACKGROUND: Although the plant microbiome is crucial for plant health, little is known about the significance of the seed microbiome. Here, we studied indigenous bacterial communities associated with the seeds in different cultivars of oilseed rape and their interactions with symbiotic and pathogenic microorganisms. RESULTS: We found a high bacterial diversity expressed by tight bacterial co-occurrence networks within the rape seed microbiome, as identified by llumina MiSeq amplicon sequencing. In total, 8362 operational taxonomic units (OTUs) of 40 bacterial phyla with a predominance of Proteobacteria (56%) were found. The three cultivars that were analyzed shared only one third of the OTUs. The shared core of OTUs consisted mainly of Alphaproteobacteria (33%). Each cultivar was characterized by having its own unique bacterial structure, diversity, and proportion of unique microorganisms (25%). The cultivar with the lowest bacterial abundance, diversity, and the highest predicted bacterial metabolic activity rate contained the highest abundance of potential pathogens within the seed. This data corresponded with the observation that seedlings belonging to this cultivar responded more strongly to the seed treatments with bacterial inoculants than other cultivars. Cultivars containing higher indigenous diversity were characterized as having a higher colonization resistance against beneficial and pathogenic microorganisms. Our results were confirmed by microscopic images of the seed microbiota. CONCLUSIONS: The structure of the seed microbiome is an important factor in the development of colonization resistance against pathogens. It also has a strong influence on the response of seedlings to biological seed treatments. These novel insights into seed microbiome structure will enable the development of next generation strategies combining both biocontrol and breeding approaches to address world agricultural challenges.

Current Insights into the Role of Rhizosphere Bacteria in Disease Suppressive Soils.

Disease suppressive soils offer effective protection to plants against infection by soil-borne pathogens, including fungi, oomycetes, bacteria, and nematodes. The specific disease suppression that operates in these soils is, in most cases, microbial in origin. Therefore, suppressive soils are considered as a rich resource for the discovery of beneficial microorganisms with novel antimicrobial and other plant protective traits. To date, several microbial genera have been proposed as key players in disease suppressiveness of soils, but the complexity of the microbial interactions as well as the underlying mechanisms and microbial traits remain elusive for most disease suppressive soils. Recent developments in next generation sequencing and other 'omics' technologies have provided new insights into the microbial ecology of disease suppressive soils and the identification of microbial consortia and traits involved in disease suppressiveness. Here, we review the results of recent 'omics'-based studies on the microbial basis of disease suppressive soils, with specific emphasis on the role of rhizosphere bacteria in this intriguing microbiological phenomenon.

Diversity and Activity of Lysobacter Species from Disease Suppressive Soils.

The genus Lysobacter includes several species that produce a range of extracellular enzymes and other metabolites with activity against bacteria, fungi, oomycetes, and nematodes. Lysobacter species were found to be more abundant in soil suppressive against the fungal root pathogen Rhizoctonia solani, but their actual role in disease suppression is still unclear. Here, the antifungal and plant growth-promoting activities of 18 Lysobacter strains, including 11 strains from Rhizoctonia-suppressive soils, were studied both in vitro and in vivo. Based on 16S rRNA sequencing, the Lysobacter strains from the Rhizoctonia-suppressive soil belonged to the four species Lysobacter antibioticus, Lysobacter capsici, Lysobacter enzymogenes, and Lysobacter gummosus. Most strains showed strong in vitro activity against R. solani and several other pathogens, including Pythium ultimum, Aspergillus niger, Fusarium oxysporum, and Xanthomonas campestris. When the Lysobacter strains were introduced into soil, however, no significant and consistent suppression of R. solani damping-off disease of sugar beet and cauliflower was observed. Subsequent bioassays further revealed that none of the Lysobacter strains was able to promote growth of sugar beet, cauliflower, onion, and Arabidopsis thaliana, either directly or via volatile compounds. The lack of in vivo activity is most likely attributed to poor colonization of the rhizosphere by the introduced Lysobacter strains. In conclusion, our results demonstrated that Lysobacter species have strong antagonistic activities against a range of pathogens, making them an important source for putative new enzymes and antimicrobial compounds. However, their potential role in R. solani disease suppressive soil could not be confirmed. In-depth omics'-based analyses will be needed to shed more light on the potential contribution of Lysobacter species to the collective activities of microbial consortia in disease suppressive soils.

Comparative genomics and metabolic profiling of the genus Lysobacter.

BACKGROUND: Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses. RESULTS: Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani. CONCLUSIONS: Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.

Indigenous populations of three closely related Lysobacter spp. in agricultural soils using real-time PCR.

Previous research had shown that three closely related species of Lysobacter, i.e., Lysobacter antibioticus, Lysobacter capsici, and Lysobacter gummosus, were present in different Rhizoctonia-suppressive soils. However, the population dynamics of these three Lysobacter spp. in different habitats remains unknown. Therefore, a specific primer-probe combination was designed for the combined quantification of these three Lysobacter spp. using TaqMan. Strains of the three target species were efficiently detected with TaqMan, whereas related non-target strains of Lysobacter enzymogenes and Xanthomonas campestris were not or only weakly amplified. Indigenous Lysobacter populations were analyzed in soils of 10 organic farms in the Netherlands during three subsequent years with TaqMan. These soils differed in soil characteristics and crop rotation. Additionally, Lysobacter populations in rhizosphere and bulk soil of different crops on one of these farms were studied. In acid sandy soils low Lysobacter populations were present, whereas pH neutral clay soils contained high populations (respectively, <4.0-5.87 and 6.22-6.95 log gene copy numbers g(-1) soil). Clay content, pH and C/N ratio, but not organic matter content in soil, correlated with higher Lysobacter populations. Unexpectedly, different crops did not significantly influence population size of the three Lysobacter spp. and their populations were barely higher in rhizosphere than in bulk soil.

Removal of phytotoxic compounds from torrefied grass fibres by plant-beneficial microorganisms.

We aimed to select microorganisms colonizing torrefied grass fibres (TGF) and simultaneously reducing the phytotoxicity which appeared after heat treatment of the fibres. Eighty-eight bacterial strains and one fungus, previously isolated from a sequential enrichment experiment on torrefied fibres and extracts, were tested separately for their capacity to decrease phytotoxicity. Eleven of the bacterial strains and the fungus significantly reduced phytotoxicity. These organisms were checked for their ability to grow on agar containing phenol, 2-methoxyphenol, 2,6-dimethoxyphenol, 2-furalaldehyde, pyrrole-2-carboxaldehyde and furan-2-methanol as sole carbon sources. The fungus F/TGF15 and the bacterial strain 66/TGF15 were able to grow on all six compounds. Strains 15/TGE5, 23/TGE5, 43/TGE20, 56/TGF10 and 95/TGF15 grew on two to four compounds, and strain 72/TGF15 only on one compound. Strains 31/TGE5, 34/TGE5, 48/TGE20 and 70/TGF15 did not grow on any of the single toxic compounds. GC analyses of torrefied grass extracts (TGE) determined which compounds were removed by the microorganisms. F/TGF15 was the only isolate depleting phenol, 2-methoxyphenol, 2-dihydrofuranone and pyrrole-2,5-dione-3-ethyl-4-methyl. Strains 15/TGE5, 23/TGE5, 31/TGE5 and 56/TGF10, and the fungus depleted 2-furalaldehyde, 2-furan-carboxaldehyde-5-methyl, pyrrole-2-carboxaldehyde, 5-acetoxymethyl-2-furaldehyde and benzaldehyde-3-hydroxy-4-methoxy. These promising candidates for colonizing and simultaneously reducing the phytotoxicity of TGF were affiliated with Pseudomonas putida, Serratia plymuthica, Pseudomonas corrugata, Methylobacterium radiotolerans and Coniochaeta ligniaria.

Characterization of the Microbial Community Involved in the Suppression of Pythium aphanidermatum in Cucumber Grown on Rockwool.

ABSTRACT The root pathogen Pythium aphanidermatum induced lower levels of disease in cucumber (Cucumis sativus) plants on unsterilized, re-used rockwool slabs than on heat-sterilized, re-used rockwool. Several recolonization treatments of the sterilized rockwool enhanced the suppressiveness of the rockwool. Microbial community structures in the different rockwool treatments were investigated by plate counts on selective media. Disease suppressiveness in the different rockwool treatments showed the highest correlation with the culturable number of filamentous actinomycetes in both experiments (r = 0.79 and 0.94), whereas the numbers of Trichoderma spp. correlated with suppression only in the first experiment (0.86). The numbers of total culturable bacteria, fluorescent pseudomonads, Bacillus spores, and fungi all showed lower correlations with disease suppressiveness. The filamentous actinomycetes enumerated with the plate counts were mainly Streptomyces spp., of which 10% were antagonistic toward P. aphanidermatum in dual culture. The composition of the bacterial and actinomycete populations was studied with polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Multivariate analyses of these patterns with canonical correspondence analysis showed significant correlations between the microbial composition and the disease suppressiveness. However, none of the bands in PCR-DGGE patterns occurred exclusively in the treatments that had enhanced disease suppressiveness. Bands extracted from the actinomycete-specific DGGE gels showed closest similarity with members of several actinomycete genera, i.e., Streptomyces, Mycobacterium, Microbacterium, Rhodococcus, Curtobacterium, and Tsukamurella. The possible mechanism of disease suppressiveness in used rockwool slabs, based on the results obtained with culture-dependent and culture-independent detection methods, is discussed.

Characterisation of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8, a powerful antagonist of fungal diseases of cucumber.

Isolate 3.1T8 of Lysobacter enzymogenes (Christensen and Cook 1978), originating from the rhizosphere of cucumber and shown to have the potential to control Pythium aphanidermatum, is described. The strain produces extracellular proteases and lipases and shows high levels of resistance against streptomycin, kanamycin and tetracycline, but not to chloramphenicol. It shows strong in vitro antibiosis against P. aphanidermatum and several other phytopathogenic fungi. In order to identify the isolate, a carbon substrate oxidation profile (Biolog) was generated, and fatty acid methyl ester (FAME) analysis was performed. Also, the 16S rRNA gene was cloned and sequenced. With Biolog and FAME analysis, no assignment to species level was possible, because the species was not in the respective databases. BLAST analysis of the obtained sequence, followed by phylogenetic analysis, using a number of related and unrelated sequences, showed that the isolate was most closely related to Lysobacter enzymogenes (Christensen and Cook 1978).