Tapping into the maize root microbiome to identify bacteria that promote growth under chilling conditions

Background When maize (Zea mays L.) is grown in the Northern hemisphere, its development is heavily arrested by chilling temperatures, especially at the juvenile phase. As some endophytes are beneficial for plants under stress conditions, we analyzed the impact of chilling temperatures on the root microbiome and examined whether microbiome-based analysis might help to identify bacterial strains that could promote growth under these temperatures. Results We investigated how the maize root microbiome composition changed by means of 16S rRNA gene amplicon sequencing when maize was grown at chilling temperatures in comparison to ambient temperatures by repeatedly cultivating maize in field soil. We identified 12 abundant and enriched bacterial families that colonize maize roots, consisting of bacteria recruited from the soil, whereas seed-derived endophytes were lowly represented. Chilling temperatures modified the root microbiome composition only slightly, but significantly. An enrichment of several chilling-responsive families was detected, of which the Comamonadaceae and the Pseudomonadaceae were the most abundant in the root endosphere of maize grown under chilling conditions, whereas only three were strongly depleted, among which the Streptomycetaceae. Additionally, a collection of bacterial strains isolated from maize roots was established and a selection was screened for growth-promoting effects on juvenile maize grown under chilling temperatures. Two promising strains that promoted maize growth under chilling conditions were identified that belonged to the root endophytic bacterial families, from which the relative abundance remained unchanged by variations in the growth temperature. Conclusions Our analyses indicate that chilling temperatures affect the bacterial community composition within the maize root endosphere. We further identified two bacterial strains that boost maize growth under chilling conditions. Their identity revealed that analyzing the chilling-responsive families did not help for their identification. As both strains belong to root endosphere enriched families, visualizing and comparing the bacterial diversity in these communities might still help to identify new PGPR strains. Additionally, a strain does not necessarely need to belong to a high abundant family in the root endosphere to provoke a growth-promoting effect in chilling conditions

Chemically versus thermally processed brown shrimp shells or Chinese mitten crab as a source of chitin, nutrients or salts and as microbial stimulant in soilless strawberry cultivation

Brown shrimp (Crangon crangon) shells and Chinese mitten crab (Efiocheir sinensis) were chemically demineralized and deproteinized (denoted as M1 to M4 for the shrimp shells and MS to M7 for the Chinese mitten crab), and shrimp shells were torrefied at 200 to 300 degrees C (denoted as R200, R255, R300), and were compared with a commercially available chitin source (denoted as reference chitin). Based on their chemical characteristics, a selection of chitin sources was tested for their N mineralization capacity. The N release was high for the chemically treated shrimp shells and Chinese mitten crab, but not for the Dandled shrimp shells with or without acid treatment, indicating that treatment at 200 et or higher resulted in low N availability. Interaction with nutrients was tested in a leaching experiment with limed peat for three thermally and two chemically processed shrimp shells and the reference chitin source. The K concentrations in the leachate for the chemically treated shrimp shells and the reference chitin were lower than for limed peat during fertigation. Irreversible K retention was observed for one source of chemically treated shrimp shells, and the reference chitin. The thermally treated shrimp shells had a significantly higher net release of P. Na and CI than the treatment without chitin source. Three shrimp shell based materials (M4, R200 and 8300) and the reference chitin were tested in a greenhouse trial with strawberry at a dose of 2 g/L limed peat. A very positive and significant effect on Borryris cinerea disease suppression in the leaves was found for the reference chitin, M4 and R200 compared to the unamended control. The disease suppression of the 3 chitin sources was linked with an increase of the microbial biomass in the limed peat with 24% to 28% due to chitin decomposition and a 9-44% higher N uptake in the plants

Shifts in the rhizobiome during consecutive in planta enrichment for phosphate-solubilizing bacteria differentially affect maize P status

Summary Phosphorus (P) is despite its omnipresence in soils often unavailable for plants. Rhizobacteria able to solubilize P are therefore crucial to avoid P deficiency. Selection for phosphate‐solubilizing bacteria (PSB) is frequently done in vitro; however, rhizosphere competence is herein overlooked. Therefore, we developed an in planta enrichment concept enabling simultaneous microbial selection for P‐solubilization and rhizosphere competence. We used an ecologically relevant combination of iron‐ and aluminium phosphate to select for PSB in maize (Zea mays L.). In each consecutive enrichment, plant roots were inoculated with rhizobacterial suspensions from plants that had grown in substrate with insoluble P. To assess the plants’ P statuses, non‐destructive multispectral imaging was used for quantifying anthocyanins, a proxy for maize’s P status. After the third consecutive enrichment, plants supplied with insoluble P and inoculated with rhizobacterial suspensions showed a P status similar to plants supplied with soluble P. A parallel metabarcoding approach uncovered that the improved P status in the third enrichment coincided with a shift in the rhizobiome towards bacteria with plant growth‐promoting and P‐solubilizing capacities. Finally, further consecutive enrichment led to a functional relapse hallmarked by plants with a low P status and a second shift in the rhizobiome at the level of Azospirillaceae and Rhizobiaceae

Grow - store - steam - re-peat : reuse of spent growing media for circular cultivation of Chrysanthemum

Substantially extending the life span of peat- and perlite-based growing media is a measure to increase the sustainability of soilless cultivation. The extraction of peat from pristine peatlands threatens these sensitive ecosystems and carbon sinks, meanwhile resulting in increased emissions of greenhouse gasses. Each batch of peat that is reused, results in a clear reduction in CO2 emissions and a lower impact on the climate. After using growing media for one cultivation, we aim at reusing the spent material as growing medium for another crop. Spent peat and perlite-based growing media from strawberry and cucumber cultivation were upcycled after steam treatment. We tested the effectiveness of steaming to reduce phytosanitary risks. The hygienisation efficiency of the steam treatment was confirmed: plant pathogenic fungi, larvae of vine weevils and weed seeds added or already present before the process were killed by the steam treatment. As the upcycled spent growing medium already contained high nutrient levels, the fertilizer application in the reused growing medium should be reduced, especially for P and K. Five indicators for assessing stability of the materials were used: CO2 flux measurements, oxygen uptake rate (OUR), biodegradation potential, mineral N content and risk for N immobilization. The spent growing media had a low decomposition rate and the release of nutrients in a leaching experiment was lower than for a fertilized peat-based growing medium, being a reference blend for open field cultivated Chrysanthemum. N mineralisation and P uptake were tested in an incubation and pot trial, respectively, and the upcycled spent growing medium was found to be an important source of plant-available K and P. Steam treatment did not severely affect the microbial biomass and diversity of the spent growing media. Blending the steam-treated spent media with other materials or inoculating by a commercially available biocontrol fungus also had a limited effect, indicating that newly introduced microorganisms do not easily establish in steamed-treated spent growing media (SSGM). Acidification of the SSGM was achieved by a low dose of elemental S. The steam-treated growing medium was tested for growing Chrysanthemum cuttings and plantlets. Spent growing media were not able to supply sufficient mineral N, but the stored amounts of P and K in the media were sufficiently plant available for optimal crop growth

Daring to be differential : metabarcoding analysis of soil and plant-related microbial communities using amplicon sequence variants and operational taxonomical units

Background: Microorganisms are not only indispensable to ecosystem functioning, they are also keystones for emerging technologies. In the last 15 years, the number of studies on environmental microbial communities has increased exponentially due to advances in sequencing technologies, but the large amount of data generated remains difficult to analyze and interpret. Recently, metabarcoding analysis has shifted from clustering reads using Operational Taxonomical Units (OTUs) to Amplicon Sequence Variants (ASVs). Differences between these methods can seriously affect the biological interpretation of metabarcoding data, especially in ecosystems with high microbial diversity, as the methods are benchmarked based on low diversity datasets. Results: In this work we have thoroughly examined the differences in community diversity, structure, and complexity between the OTU and ASV methods. We have examined culture-based mock and simulated datasets as well as soil- and plant-associated bacterial and fungal environmental communities. Four key findings were revealed. First, analysis of microbial datasets at family level guaranteed both consistency and adequate coverage when using either method. Second, the performance of both methods used are related to community diversity and sample sequencing depth. Third, differences in the method used affected sample diversity and number of detected differentially abundant families upon treatment; this may lead researchers to draw different biological conclusions. Fourth, the observed differences can mostly be attributed to low abundant (relative abundance < 0.1%) families, thus extra care is recommended when studying rare species using metabarcoding. The ASV method used outperformed the adopted OTU method concerning community diversity, especially for fungus-related sequences, but only when the sequencing depth was sufficient to capture the community complexity. Conclusions: Investigation of metabarcoding data should be done with care. Correct biological interpretation depends on several factors, including in-depth sequencing of the samples, choice of the most appropriate filtering strategy for the specific research goal, and use of family level for data clustering

Soil under stress : the importance of soil life and how it is influenced by (micro)plastic pollution

Soil organisms and specifically microorganisms are indispensable to life on Earth. They regulate essential ecosystem functions from carbon sequestration to primary production. These organisms often experience stress when the balance of the soil system is disrupted by agricultural practices and environmental disturbances. A new stressor is plastic, which can be found in soils, in and around soil-dwelling organisms, and close to plants. The presence of plastic can affect soil chemistry, plant growth and the survival of higher-order organisms. Microbial organisms respond sensitively to these changes in their surroundings and will thus be (in)directly affected by plastic. Eventually, this results in a different microbial activity, composition and reduced diversity. Plastic might even serve as a specific habitat for microorganisms, generally referred to as the plastisphere. In this review, we make predictions based on the observed effects of (micro)plastics and the potential impact on the plant-soil-microbiome system. We use prior knowledge of other distur-bances (e.g. tillage and pesticides) which have been studied for many years in relation to the soil microbial community. Further research is needed to develop standardized methods to study smaller plastic particles (micro- and nanoplastics) as these play the most dominant role in terrestrial ecosystems

Biochar-enhanced resistance to Botrytis cinerea in strawberry fruits (but not leaves) is associated with changes in the rhizosphere microbiome

Biochar has been reported to play a positive role in disease suppression against airborne pathogens in plants. The mechanisms behind this positive trait are not well-understood. In this study, we hypothesized that the attraction of plant growth-promoting rhizobacteria (PGPR) or fungi (PGPF) underlies the mechanism of biochar in plant protection. The attraction of PGPR and PGPF may either activate the innate immune system of plants or help the plants with nutrient uptake. We studied the effect of biochar in peat substrate (PS) on the susceptibility of strawberry, both on leaves and fruits, against the airborne fungal pathogen Botrytis cinerea. Biochar had a positive impact on the resistance of strawberry fruits but not the plant leaves. On leaves, the infection was more severe compared with plants without biochar in the PS. The different effects on fruits and plant leaves may indicate a trade-off between plant parts. Future studies should focus on monitoring gene expression and metabolites of strawberry fruits to investigate this potential trade-off effect. A change in the microbial community in the rhizosphere was also observed, with increased fungal diversity and higher abundances of amplicon sequence variants classified into Granulicella, Mucilaginibacter, and Byssochlamys surrounding the plant root, where the latter two were reported as biocontrol agents. The change in the microbial community was not correlated with a change in nutrient uptake by the plant in either the leaves or the fruits. A decrease in the defense gene expression in the leaves was observed. In conclusion, the decreased infection of B. cinerea in strawberry fruits mediated by the addition of biochar in the PS is most likely regulated by the changes in the microbial community

Identification of microbial life in sustainable and disease suppressive growing media : the role of beneficial microorganisms

Our current understanding of the microbial communities inhabiting growing media is limited. However, techniques such as phospholipid fatty acid analysis, metabarcoding and shotgun metagenomics are increasingly used to analyze and understand microbial life in growing media and therefore they are starting to fill the knowledge gap. Using these techniques in the interreg2seas project Horti-BlueC (www.horti-bluec.eu ), we try to understand the microbiological processes involved in sustainable growing media based on plant fibers, chitin and biochar. In sustainable growing media, peat is (partially) replaced and/or the use of chemical fertilizers and plant protection products is reduced. Plant fibers include defibrated miscanthus straw, flax shives and reed. Chitin is produced from shell fish waste such as crab and shrimps shells. Biochar (charred material) is rich in carbon and is produced from organic material such as wood, spent growing media and the woody fraction of green waste. We showed that adding biochar to peat mainly changes the bacterial community, whereas plant fibers and chitin mainly change the fungal community of the growing media. By changing the microbiome of the growing media, these new amendments might reduce the impact of horticulture on the environment and contribute to a circular-based economy

Has compost with biochar applied during the process added value over biochar or compost for increasing soil quality in an arable cropping system?

Previous research showed that adding biochar at the onset of the composting process alleviates this process and reduces emissions. The objective of this study was to evaluate in a field trial whether this compost with biochar applied during the process (i.e., biochar-blended compost) has added value compared to compost or biochar alone on chemical, physical and biological soil properties and crop yield. A single application of biochar, compost and biochar-blended (BB) compost increased the C content of the top soil in the long term, but only compost and biochar-blended compost had a lasting effect on pH and the K content. Two to 4 years after a single application of all three amendments in the field, the soil microbial biomass, richness, diversity and community composition remained unchanged. Due to intensive soil tillage, biochar migrated to subsoil already after 2 growing seasons, thus increasing the C sequestration in subsoil. We conclude that in general, the biochar-blended compost outperformed biochar and had a similar effect as the compost