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.

Species Identity, Life History, and Geographic Distance Influence Gut Bacterial Communities in Lab-Reared and European Field-Collected Culicoides Biting midges.

Bacteria are part of the insect gut system and influence many physiological traits of their host. Gut bacteria may even reduce or block the transmission of arboviruses in several species of arthropod vectors. Culicoides biting midges are important arboviral vectors of several livestock and wildlife diseases, yet limited information is available on their gut bacterial communities. Addressing this gap will help inform how these communities can be manipulated and ultimately used as novel tools to control pathogens. To assess how bacterial communities change during the life stages of lab-reared C. nubeculosus and C. sonorensis, endosymbiotic bacteria were identified using Illumina sequencing of 16S rRNA and taxonomically characterised. Analyses were conducted to determine how gut bacterial communities in adults are influenced by species identity and geographic distance among biting midge populations. Communities of the two lab-reared Culicoides species significantly changed after pupation and with maturation into 6-day-old adults. Pseudomonas, Burkholderiaceae and Leucobacter bacteria were part of a core community that was trans-stadially transmitted and found throughout their life cycle. Among field-collected biting midges, the bacterial communities were unique for almost each species. Cardinium, Rickettsia and Wolbachia were some of the most abundant bacteria in midges collected from wetlands. Only Pseudomonas was present in high relative abundance in all field-collected species. In this study, species identity, as well as geographic distance, influenced the gut bacterial communities and may partly explain known inter- and intra-species variability in vector competence. Additionally, stably associated bacterial species could be candidates for paratransgenic strategies to control vector-borne pathogens.

Transmission of Escherichia coli from Manure to Root Zones of Field-Grown Lettuce and Leek Plants.

Pathogenic Escherichia coli strains are responsible for food-borne disease outbreaks upon consumption of fresh vegetables and fruits. The aim of this study was to establish the transmission route of E. coli strain 0611, as proxy for human pathogenic E. coli, via manure, soil and plant root zones to the above-soil plant compartments. The ecological behavior of the introduced strain was established by making use of a combination of cultivation-based and molecular targeted and untargeted approaches. Strain 0611 CFUs and specific molecular targets were detected in the root zones of lettuce and leek plants, even up to 272 days after planting in the case of leek plants. However, no strain 0611 colonies were detected in leek leaves, and only in one occasion a single colony was found in lettuce leaves. Therefore, it was concluded that transmission of E. coli via manure is not the principal contamination route to the edible parts of both plant species grown under field conditions in this study. Strain 0611 was shown to accumulate in root zones of both species and metagenomic reads of this strain were retrieved from the lettuce rhizosphere soil metagenome library at a level of Log 4.11 CFU per g dry soil.

Log-ratio analysis of microbiome data with many zeroes is library size dependent.

Microbiome composition data collected through amplicon sequencing are count data on taxa in which the total count per sample (the library size) is an artefact of the sequencing platform, and as a result, such data are compositional. To avoid library size dependency, one common way of analysing multivariate compositional data is to perform a principal component analysis (PCA) on data transformed with the centred log-ratio, hereafter called a log-ratio PCA. Two aspects typical of amplicon sequencing data are the large differences in library size and the large number of zeroes. In this study, we show on real data and by simulation that, applied to data that combine these two aspects, log-ratio PCA is nevertheless heavily dependent on the library size. This leads to a reduction in power when testing against any explanatory variable in log-ratio redundancy analysis. If there is additionally a correlation between the library size and the explanatory variable, then the type 1 error becomes inflated. We explore putative solutions to this problem.

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.

Impact of Gut Bacteria on the Infection and Transmission of Pathogenic Arboviruses by Biting Midges and Mosquitoes.

Tripartite interactions among insect vectors, midgut bacteria, and viruses may determine the ability of insects to transmit pathogenic arboviruses. Here, we investigated the impact of gut bacteria on the susceptibility of Culicoides nubeculosus and Culicoides sonorensis biting midges for Schmallenberg virus, and of Aedes aegypti mosquitoes for Zika and chikungunya viruses. Gut bacteria were manipulated by treating the adult insects with antibiotics. The gut bacterial communities were investigated using Illumina MiSeq sequencing of 16S rRNA, and susceptibility to arbovirus infection was tested by feeding insects with an infectious blood meal. Antibiotic treatment led to changes in gut bacteria for all insects. Interestingly, the gut bacterial composition of untreated Ae. aegypti and C. nubeculosus showed Asaia as the dominant genus, which was drastically reduced after antibiotic treatment. Furthermore, antibiotic treatment resulted in relatively more Delftia bacteria in both biting midge species, but not in mosquitoes. Antibiotic treatment and subsequent changes in gut bacterial communities were associated with a significant, 1.8-fold increased infection rate of C. nubeculosus with Schmallenberg virus, but not for C. sonorensis. We did not find any changes in infection rates for Ae. aegypti mosquitoes with Zika or chikungunya virus. We conclude that resident gut bacteria may dampen arbovirus transmission in biting midges, but not so in mosquitoes. Use of antimicrobial compounds at livestock farms might therefore have an unexpected contradictory effect on the health of animals, by increasing the transmission of viral pathogens by biting midges.

Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum).

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.

Bacterial communities associated with Chenopodium album and Stellaria media seeds from arable soils.

The bacterial community compositions in Chenopodium album and Stellaria media seeds recovered from soil (soil weed seedbank), from bulk soil, and from seeds harvested from plants grown in the same soils were compared. It was hypothesized that bacterial communities in soil weed seedbanks are distinct from the ones present in bulk soils. For that purpose, bacterial polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints, made from DNA extracts of different soils and seed fractions, were analyzed by principal component analysis. Bacterial fingerprints from C. album and S. media seeds differed from each other and from soil. Further, it revealed that bacterial fingerprints from soil-recovered and plant-harvested seeds from the same species clustered together. Hence, it was concluded that microbial communities associated with seeds in soil mostly originated from the mother plant and not from soil. In addition, the results indicated that the presence of a weed seedbank in arable soils can increase soil microbial diversity. Thus, a change in species composition or size of the soil weed seedbank, for instance, as a result of a change in crop management, could affect soil microbial diversity. The consequence of increased diversity is yet unknown, but by virtue of identification of dominant bands in PCR-DGGE fingerprints as Lysobacter oryzae (among four other species), it became clear that bacteria potentially antagonizing phytopathogens dominate in C. album seeds in soil. The role of these potential antagonists on weed and crop plant growth was discussed.