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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., Sequential in planta enrichment for phosphate solubilizing bacteria led to plants releaved of P‐stress, hallmarked by reduced anthocyanin accumulation and a shift in the rhizobiome.

Strawberry cultivation is associated with high mineral fertilizer doses and extensive use of chemical plant protection products. Based on previous research, we expected that chitin application to peat substrate would increase the nutrient availability and activate the plant systemic defense response, resulting in higher strawberry yields and fewer disease symptoms. We set up two experiments in which the temporal variability and differences in initial nutrient concentrations of the growing media were taken into account. Chitin treatment resulted in the attraction of plant growth–promoting fungi toward the plant root, such as species from genera Mortierella and Umbelopsis. In addition, by the end of the experiments 87 mg of mineral nitrogen (N) per liter of substrate was mineralized, which can be related to the observed increase in plant shoot biomass. This, however, led to nutrient imbalances in plant shoots and fruit; N concentration in the leaves increased over 30%, exceeding the optimal range, while phosphorous (P) and potassium (K) deficiencies occurred, with concentrations lower than 50% of the optimal range. This may explain the decreased fruit yield and disease resistance of the fruit toward Botrytis cinerea. In contrast, chitin caused a clear defense priming effect in the strawberry leaves, with a strong induction of the jasmonic acid response, resulting in fewer foliar disease symptoms. Chitin causes positive effects on shoot growth and foliar disease resistance, but caution needs to be taken for nutrient imbalances leading to negative influences on root growth, fruit production, and disease susceptibility toward B. cinerea. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

This review summarizes the most common biological methods and their conditions for the recovery of bioactive compounds such as chitin/chitosan, proteins/peptides and carotenoids from marine crustacean processing side-streams (MCPS). Chitin can be efficiently extracted from MCPS by adding proteases to hydrolyze the proteins (enzymatic hydrolysis), or by using (non-)lactic acid fermentation to digest and eliminate nonchitinous compounds. Peptides with antimicrobial, antioxidant and antihypertensive activities can be generated via fermentation and enzymatic hydrolysis; in addition, carotenoid extraction can be enhanced by both techniques. This review also briefly demonstrates the advantages, challenges and future perspectives of applying biological methods for the valorization of MCPS. These biological methods may provide an alternative solution to minimize the environmental and economic impact of inefficient waste management. Moreover, MCPS can be used to produce high value-added compounds with potent bioactivities. Ultimately, these compounds produced via optimized biological methods can be commercialized for different applications. (c) 2021 Society of Chemical Industry

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 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.

Trichoderma spp. are one of the most commercialized fungal biocontrol agents. To improve its applicability, while preserving its effectiveness against diseases, a selection of composts were tested as carrier mediums. The population dynamic of T. harzianum was studied in three composts differing in biological characteristics based on the Nematode Index of Compost Maturity (NICM). The results showed a decline in the T. harzianum population, which became stable after six or eight weeks, irrespective of compost, concentration or sterilization. The bioassay with Rhizoctonia solani on bean showed promising results for disease control after storage of the inoculated composts. Nevertheless, T. harzianum was ineffective against R. solani in one compost with a high NICM value, likely related to its sub-optimal physicochemical characteristics and lower root colonization. Compost is a suitable carrier medium for T. harzianum, but the NICM value is not a good indicator to predict the survival rate nor the disease suppression activity of compost inoculated with T. harzianum.

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.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter. Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize (Zea mays), is investigated in Arabidopsis thaliana. RHG1 colonized both roots and shoots of Arabidopsis, promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.

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.

Three characteristics are considered key for optimal use of composts in growing media: maturity, pH and organic matter content. Maturation is a critical step in the processing of composts contributing to compost quality. Blending of composts with chopped heath biomass, sieving out the larger fraction of composts and acidification of composts by adding elemental sulfur may be used either to increase organic matter content or to reduce pH for a better fit in growing media. While several studies have shown the effectiveness of these treatments to improve the use of composts in growing media, the effect of these treatments on the compost microbiome has merely been assessed before. In the present study, five immature composts were allowed to mature, and were subsequently acidified, blended or sieved. Bacterial and fungal communities of the composts were characterized and quantified using 16S rRNA and ITS2 gene metabarcoding and phospholipid fatty acid analysis. Metabolic biodiversity and activity were analyzed using Biolog EcoPlates. Compost batch was shown to be more important than maturation or optimization treatments to determine the compost microbiome. Compost maturation increased microbial diversity and favored beneficial microorganisms, which may be positive for the use of composts in growing media. Blending of composts increased microbial diversity, metabolic diversity, and metabolic activity, which may have a positive effect in growing media. Blending may be used to modify the microbiome to a certain degree in order to optimize microbiological characteristics. Acidification caused a decrease in bacterial diversity and microbial activity, which may be negative for the use in growing media, although the changes are limited. Sieving had limited effect on the microbiome of composts. Because of the limited effect on the microbiome, sieving of composts may be used flexible to improve (bio)chemical characteristics. This is the first study to assess the effects of maturation and optimization treatments to either increase organic matter content or lower pH in composts on the compost microbiome. Bi-o-ptimal@work (Sustainable cultivation in container and open field by using innovative and local materials with enhanced microbial life, ready for use and implementation by ornamental growers) is funded by Flanders Innovation and Entrepreneurship, project number HBC.2017.0815. CD received a grant of the Research Foundation Flanders (FWO) with application number 12S9418N.

While many phenylpropanoid pathway-derived molecules act as physical and chemical barriers to pests and pathogens, comparatively little is known about their role in regulating plant immunity. To explore this research field, we transiently perturbed the phenylpropanoid pathway through application of the CINNAMIC ACID-4-HYDROXYLASE (C4H) inhibitor piperonylic acid (PA). Using bioassays involving diverse pests and pathogens, we show that transient C4H inhibition triggers systemic, broad-spectrum resistance in higher plant without affecting growth. PA treatment enhances tomato (Solanum lycopersicum) resistance in field and laboratory conditions, thereby illustrating the potential of phenylpropanoid pathway perturbation in crop protection. At the molecular level, transcriptome and metabolome analyses reveal that transient C4H inhibition in tomato reprograms phenylpropanoid and flavonoid metabolism, systemically induces immune signaling and pathogenesis-related genes, and locally affects reactive oxygen species metabolism. Furthermore, C4H inhibition primes cell wall modification and phenolic compound accumulation in response to root-knot nematode infection. Although PA treatment induces local accumulation of the phytohormone salicylic acid, the PA resistance phenotype is preserved in tomato plants expressing the salicylic acid-degrading NahG construct. Together, our results demonstrate that transient phenylpropanoid pathway perturbation is a conserved inducer of plant resistance and thus highlight the crucial regulatory role of this pathway in plant immunity.

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.

Plant fibers allow for the partial replacement of peat in growing media and have potential to carry and sustain biocontrol organisms. However, they may also carry plant pathogens and they may vary in their degree of biodegradability and their interaction with N, and in this way thus affect the quality of growing media. We tested 3 types of plant fibers (flax, miscanthus and reed) for replacement of peat in growing media. In order to (1) kill plant pathogens present on the plant fibers, (2) reduce the N fixation risk of the fibers and (3) provide fibers serving as carrier of biocontrol fungi, various defibration techniques were tested, including extrusion, retruding, disc refining and steam explosion. Flax shives obtained from various sources were naturally colonized by viable microsclerotia of the plant pathogen Verticillium dahliae. When sufficiently high temperatures were reached during defibration, this technique killed the plant pathogen on the shives. Untreated flax shives, miscanthus or reed straw were characterized by high N immobilization and thus reduce N availability for plants if used as growing medium. We found a clear positive correlation between the risk for N immobilization versus pH, the hemicellulose content and water-extractable C concentration of the fiber. Unlike peat, defibrated pure miscanthus, reed straw and flax shives were easily colonized by fungal biocontrol strains. Inoculation of extruded miscanthus fibers with biocontrol fungi did not increase nor decrease the N immobilization. Reducing the pH of the fibers was effective in reducing the microbial activity and the N fixation. Defibrated plant fibers have potential for peat replacement and as carrier for biocontrol fungi. Peat replacement is important in relation to the circular economy, climate change mitigation (prevention of carbon loss and greenhouse gas emissions) and the conservation of fragile ecosystems. Successful application of biocontrol fungi may reduce the need for using chemical crop protection agents in horticulture.

Several materials to be used in growing media were tested for immobilization of mineral N by adding 350 mg N L-1 substrate as KNO3 followed by one week of incubation at 37°C. Based on the difference between the theoretical and actual mineral N content after incubation, the % N immobilization was calculated. The robustness and reproducibility of the protocol was tested by changing incubation circumstances, the applied N sources and the amount of N added. The relevance of the indicator was tested (a) for a range of green waste composts, (b) during the composting of four mixtures, and (c) in pot trials with growing media including different composts and lignocellulosic materials. Higher microbial activity in green waste composts (indicated by higher oxygen consumption) and thus a lower compost stability resulted in higher N immobilization. N immobilization decreased due to stabilization during the composting process, with a distinct effect of the specific feedstock composition. The protocol was tested for woody materials and for Miscanthus straw, spent coffee grounds and chopped reed straw. The applied source of mineral N in the test affected the pH of the material, and may thus also affect the microbial activity and thus the related N immobilisation. Growing media including materials with high N immobilization resulted in lower plant growth. We conclude that quality of composts and lignocellulosic materials for use in growing media could be successfully assessed based on this protocol.

Chitin is a valuable peat substrate amendment by increasing lettuce growth and reducing the survival of the zoonotic pathogen Salmonella enterica on lettuce leaves. The production of chitin-catabolic enzymes (chitinases) play a crucial role and are mediated through the microbial community. A higher abundance of plant-growth promoting microorganisms and genera involved in N and chitin metabolism are present in a chitin-enriched substrate. In this study, we hypothesize that chitin addition to peat substrate stimulates the microbial chitinase production. The degradation of chitin leads to nutrient release and the production of small chitin oligomers that are related to plant growth promotion and activation of the plant’s defense response. First a shotgun metagenomics approach was used to decipher the potential rhizosphere microbial functions then the nutritional content of the peat substrate was measured. Our results show that chitin addition increases chitin-catabolic enzymes, bacterial ammonium oxidizing and siderophore genes. Lettuce growth promotion can be explained by a cascade degradation of chitin to N-acetylglucosamine and eventually ammonium. The occurrence of increased ammonium oxidizing bacteria, Nitrosospira, and amoA genes results in an elevated concentration of plant-available nitrate. In addition, the increase in chitinase and siderophore genes may have stimulated the plant’s systemic resistance.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are major constraints to potato crop production. We studied the effects of several soil amendments on PCN survival and reproduction in pot experiments. Pig slurry, cattle slurry, mineral nitrogen fertilizer (NH4NO3), crab shell compost and wood chip compost at 170 kg N ha−1 reduced the number of viable eggs in cysts of both PCN species in the absence of potato. This resulted in fewer second-stage juveniles (J2) hatching from these cysts and penetrating potato roots than from cysts of non-amended soils. When potato was planted, the same amendments resulted in less reproduction than in non-amended soil. Most reduction of reproduction was achieved in soils amended with pig slurry (87%) and wood chip compost (82%). Adding biochar at 0.3 and 1% did not reduce the survival or the reproduction of any of PCN species; moreover, it inhibited the suppressing effect of wood chip compost and pig slurry on PCN reproduction when added together with these amendments. The release of ammonium and changes in soil microbial community, determined by phospholipid fatty acid (PLFA) analysis, are involved in nematode suppression in soil amended with pig and cattle slurries. However, the suppressing effect of wood chip and crab shell compost can only be explained by the changes in soil microbiota, while the effect of mineral nitrogen fertilizer can only be related with the production of ammonium. Ammonium and microorganisms most probably have affected PCN directly by killing the eggs and juveniles or indirectly by changing the physiology of the root as mentioned amendments reduced hatch and movement of J2, penetration of the roots and females’ fecundity.

Biochar, a solid coproduct of biomass pyrolysis, has recently been proposed as soil amendment in agriculture. We studied the effect of biochar on soil and substrate physicochemical properties, plant growth, disease susceptibility and rhizosphere microbiology in two contrasting cropping systems, i.e., lettuce grown in field soil and strawberry grown in white peat. For both systems, changes in the physicochemical properties of the plant growth media were observed. In the lettuce bio-assay, biochar addition had no effect on crop growth, crop health (Rhizoctonia solani infection test) and rhizosphere microbiology. In contrast, in the strawberry bioassay, addition of 3% biochar to peat resulted in (i) a higher fresh and dry plant weight, (ii) a lower susceptibility for the fungal pathogen Botrytis cinerea on both leaves and fruits, and (iii) changes in the rhizosphere microbiology, analysed by Phospholipid Fatty Acid (PLFA) profiling and 16S rDNA amplicon sequencing. Biochar addition led to an increase of bacterial diversity and a shift in composition of the rhizosphere microbiota. Extra inorganic plant nutrition and lime added to the peat reduced these effects of biochar on the strawberry plants. We conclude that in certain plant growth media, biochar amendment can result in chemical changes that induce multiple responses in the plant, including shifts in the rhizosphere microbiome. Biochar can be beneficial for plant growth, especially in conditions of limited nutrient availability.

Farmers have to combine several measures to balance carbon contents in the top soil to preserve soil quality but potentially increasing the risk for N and P losses by leaching. We studied the effect of a combination of several measures on topsoil C content, nutrient leaching and soil quality in a multi-year field trial. Soil quality was defined as the result of the interaction between chemical, physical and biological soil characteristics, and included an assessment of disease suppressiveness as well. Initially, the soil had a suboptimal C content (i.e. 0.81%). Soil organic carbon (SOC) levels were maintained and slightly increased by applying slurry and good agricultural practices such as the use of cover crops and the incorporation of cereal straw (on average +0.04 percentage point). Only with a yearly extra plant-based farm compost amendment SOC levels were substantially increased after a period of four years (on average +0.17 percentage point). This application of 8.3 Mg C ha−1 also improved chemical, physical and biological soil quality. Our research demonstrates that farmers can use compost, for at least 4 years, on top of cattle or pig slurry application to soils with suboptimal C levels to increase C content in the top soil, without inducing higher N and P leaching. Repeated compost application increased the microbial biomass (measured by means of PLFA) and disease suppressiveness against Botrytis cinerea on lettuce. There was no effect on the abundance of the plant-parasitic nematode Pratylenchus penetrans in the soil, nor on disease severity caused by Dickeya solani, Streptomyces scabies and Rhizoctonia solani after a potato cropping. Differences between tillage practices were most obvious in the 0–10 cm soil layer as non-inversion tillage (compared to ploughing) resulted in an increase in SOC, Total N, hot-water extractable C and P, plant-available P and K, aggregate stability, earthworm abundance and bacterial and fungal populations in this layer.

Adding biochar at the onset of composting has proven to be beneficial for this process, and when added to the soil, this biochar-blended (BB) compost has outperformed biochar on soil quality but had a similar effect as the compost. Because soil quality gets more and more attention as an important tool for soil health, pot trials were conducted to evaluate the effect of compost and biochar alone, and biochar-blended (BB) compost on disease suppression. In addition, it was investigated whether the disease suppression was associated with changes in soil quality (chemical, physical and biological soil properties). The two selected bio-assays were Rhizoctonia solani (basal rot) on lettuce and potato cyst nematodes (PCN) on potato. Both compost and BB compost released mineral N and thus increased lettuce and potato yield but biochar did not. None of the amendments affected the susceptibly of lettuce leaves to basal rot. Compost suppressed PCN while biochar had no effect on PCN reproduction. Moreover, BB compost reduced the suppressing effect of compost. We suggest that the inhibitory effect of biochar blended in compost might be attributed to the fungal biomass which was not increased in the BB compost-amended soil compared to compost-amended soil, and/or the absorbance of suppressive compounds by the high surface area of biochar. Interestingly, all three amendments reduced the water evaporation from the soil surface, which can have important implications for drought stress of plants.

Peat based growing media are not ecologically sustainable and often fail to support biological control. Miscanthus straw was (1) tested to partially replace peat; and (2) pre-colonized with a Trichoderma strain to increase the biological control capacity of the growing media. In two strawberry pot trials (denoted as experiment I & II), extruded and non-extruded miscanthus straw, with or without pre-colonization with T. harzianum T22, was used to partially (20% v/v) replace peat. We tested the performance of each mixture by monitoring strawberry plant development, nutrient content in the leaves and growing media, sensitivity of the fruit to the fungal pathogen Botrytis cinerea, rhizosphere community and strawberry defense responses. N immobilization by miscanthus straw reduced strawberry growth and yield in experiment II but not in I. The pre-colonization of the straw with Trichoderma increased the post-harvest disease suppressiveness against B. cinerea and changed the rhizosphere fungal microbiome in both experiments. In addition, defense-related genes were induced in experiment II. The use of miscanthus straw in growing media will reduce the demand for peat and close resource loops. Successful pre-colonization of this straw with biological control fungi will optimize crop cultivation, requiring fewer pesticide applications, which will benefit the environment and human health.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are major constraints to potato production. They are distributed passively with soil adhering to farm machinery and harvested tubers, but also by waste soil from trading and processing plants. Inundation is considered an environmentally save method for disinfestation of waste soil. We investigated if agro-industrial waste products accelerate this disinfestation process by studying PCN survival, hatching and infectivity over time. Experiments were conducted in 2-L containers filled with 1.5 L non-amended soil, soil amended with steamed or fresh potato peels, calcium sulphate, steamed potato peels mixed with calcium sulphate, or leek leaves. In each container, cysts of G. rostochiensis or G. pallida were buried in the soil in retrievable bags and 400 ml water was added. Non-amended and non-inundated soils were the controls. It took 8 weeks of inundation of non-amended soil to reduce nematode survival by 72 %, while in amended soils survival was reduced up to 99.9 % after 4 weeks. After 1 week, fewer second-stage juveniles (J2) hatched and infected roots in inundated amended soil than in inundated non-amended soil. Host finding ability of surviving J2 was not affected. To understand mechanisms of nematode suppression, concentrations of short chain fatty acids (SCFA), gases (O2, CO2, H2S, CH4, and N2) and phospholipid fatty acids (PLFAs) were determined. Higher concentrations of SCFA and carbon dioxide and lower levels of oxygen were detected in amended treatments. H2S was occasionally detected in the leek-amended soil. The microbial biomass changed significantly in potato peels-amended soils.

Adding biochar, the solid coproduct of biofuel production, to peat can enhance strawberry growth, and disease resistance against the airborne fungal pathogen Botrytis cinerea. Additionally, biochar can induce shifts in the strawberry rhizosphere microbiome. However, the moment that this biochar-mediated shift occurs in the rhizosphere is not known. Further, the effect of an above-ground infection on the strawberry rhizosphere microbiome is unknown. In the present study we established two experiments in which strawberry transplants (cv. Elsanta) were planted either in peat or in peat amended with 3% biochar. First, we established a time course experiment to measure the effect of biochar on the rhizosphere bacterial and fungal communities over time. In a second experiment, we inoculated the strawberry leaves with B. cinerea, and studied the impact of the infection on the rhizosphere bacterial community. The fungal rhizosphere community was stabilized after 1 week, except for the upcoming Auriculariales, whereas the bacterial community shifted till 6 weeks. An effect of the addition of biochar to the peat on the rhizosphere microbiome was solely measured for the bacterial community from week 6 of plant growth onwards. When scoring the plant development, biochar addition was associated with enhanced root formation, fruit production, and postharvest resistance of the fruits against B. cinerea. We hypothesize that the bacterial rhizosphere microbiome, but also biochar-mediated changes in chemical substrate composition could be involved in these events. Infection of the strawberry leaves with B. cinerea induced shifts in the bacterial rhizosphere community, with an increased bacterial richness. This disease-induced effect was not observed in the rhizospheres of the B. cinerea-infected plants grown in the biochar-amended peat. The results show that an above-ground infection has its effect on the strawberry rhizosphere microbiome, changing the bacterial interactions in the root-substrate interface. This infection effect on the bacterial rhizosphere microbiome seems to be comparable to, but less pronounced than the effect of biochar-addition to the peat. The biological meaning of these observations needs further research, but this study indicates that biochar and an above-ground pathogen attack help the plant to recruit rhizosphere microbes that may aid them in their plant growth and health.

Chitin is a promising soil amendment for improving soil quality, plant growth and plant resilience. The objectives of this study were twofold. First, to study the effect of chitin mixed in potting soil on lettuce growth and on the survival of two zoonotic bacterial pathogens, Escherichia coli O157:H7 and Salmonella enterica on the lettuce leaves. Second, to assess the related changes in the microbial lettuce rhizosphere, using phospholipid fatty acid (PLFA) analysis and amplicon sequencing of a bacterial 16S rRNA gene fragment and the fungal ITS2. As a result of chitin addition, lettuce fresh yield weight was significantly increased. S. enterica survival in the lettuce phyllosphere was significantly reduced. The E. coli O157:H7 survival was also lowered, but not significantly. Moreover, significant changes were observed in the bacterial and fungal community of the lettuce rhizosphere. PLFA analysis showed a significant increase in fungal and bacterial biomass. Amplicon sequencing showed no increase in fungal and bacterial biodiversity, but relative abundances of the bacterial phyla Acidobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, and Proteobacteria and the fungal phyla Ascomycota, Basidiomycota, and Zygomycota were significantly changed. More specifically, a more than tenfold increase was observed for operational taxonomic units (OTUs) belonging to the bacterial genera Cellvibrio, Pedobacter, Dyadobacter, and Streptomyces and to the fungal genera Lecanicillium and Mortierella. These genera include several species previously reported to be involved in biocontrol, plant growth promotion, the nitrogen cycle and chitin degradation. These results enhance the understanding of the response of the rhizosphere microbiome to chitin amendment. Moreover, this is the first study to investigate the use of soil amendments to control the survival of S. enterica on plant leaves.

Fusarium foetens is a recently described aggressive vascular pathogen of Begonia x hiemalis. Since 2004, it has caused severe losses for Begonia growers in Northern Europe and North America. F. foetens is likely to be of exotic origin. Little is known about the accumulation of the fungus in Begonia plants before and during symptom expression and about its host range. We have optimised a molecular detection method for F. foetens by only using the plant part containing the largest amount of the pathogen and by optimising the tissue maceration and DNA extraction techniques. This allowed a reliable detection limit of 2310 spore equivalents per plant and a theoretical detection limit of as low as 84 to 167 spore equivalents per plant. Using this method, we demonstrated exponential accumulation of F. foetens DNA in Begonia roots, resulting in symptoms at a threshold of approximately 107 spore equivalents and levelling off at 109 spore equivalents per plant. The observed rate of accumulation and the amount of pathogen DNA in non-symptomatic plants can be combined to determine whether the cuttings were infected after delivery at the Begonia nursery and to calculate the estimated timing of symptom development. To test the host range, we applied the optimised molecular detection technique. During these tests, only Begonia x hiemalis plants became symptomatic, but many other plant species supported growth of the pathogen. This information can be used to aid pathogen control and has implications for pest risk assessment.

Colletotrichum isolates (457) were collected from strawberry plant tissues with and without typical anthracnose symptoms and from symptomless weeds in 19 Belgian strawberry fields. The isolates were characterized based on genetic, morphological and pathological features. Isolates were classified according to rDNA-ITS sequencing: 97% of 211 representative isolates were C. acutatum, 2%C. gloeosporioides and 1%C. coccodes. The C. acutatum isolates belonged to the intraspecific groups A2 (33%), A3 (5%), A4 (50%), A5 (3%) and A7 (6%). Differences in spore morphology, growth rate and colony colour of a selection of 146 isolates confirmed the genetic grouping. Multiple Colletotrichum genotypes were detected in the same field. There was no association between the most common genotypes and geographic origin, presence or absence of symptoms, nor plant species or plant part. Representative Belgian Colletotrichum isolates were used in pathogenicity tests, together with European and American reference isolates. The C. acutatum A2 and the Belgian C. gloeosporioides isolates were the most aggressive on fruits, followed by C. acutatum A3, A4, A5, A7 and C. coccodes isolates. When inoculated into crowns, C. acutatum A2, A5 and American C. gloeosporioides isolates were the most aggressive, followed by C. acutatum A3 isolates. The A4 and A7 isolates and all European C. gloeosporioides isolates were non-pathogenic on crowns. These data indicate that an unusually diverse Colletotrichum population is present in Belgium. The traditional differentiation between C. acutatum and C. gloeosporioides as causal agents of fruit and crown rot, respectively, proved not to be valid in Belgian strawberry fields.

Aims: To study the phenotypic and genetic diversity of culturable bacteria associated with rice seed and to asses the antagonistic and pathogenic potential of the isolated bacteria. Methods and Results: Seed of rice cultivar PSBRc14 was collected from farmers’ fields of irrigated lowland in southern Luzon, Philippines. Isolations of distinct colonies yielded 498 isolates. Classification of the isolates according to similarities in cellular characteristics, whole-cell fatty acid composition, and colony appearance differentiated 101 morphotype groups. Predominant bacteria were Coryneform spp., Pantoea spp. and Pseudomonas spp. Other bacteria regularly present were Actinomycetes spp., Bacillus pumilus, B. subtilis, Burkholderia glumae, Enterobacter cloacae, Paenibacillus polymyxa, Staphylococcus spp. and Xanthomonas spp. The genetic diversity among isolates was assessed by BOX-PCR fingerprinting of genomic DNA and represented 284 fingerprint types (FPTs). Most FPTs (78%) were not shared among samples, while eight FPTs occurred frequently in the samples. Seven of these FPTs also occurred frequently in a previous collection made from rainfed lowlands of Iloilo island, Philippines. Sixteen per cent of the isolates inhibited in vitro the mycelial growth of the rice pathogens Rhizoctonia solani and Pyricularia grisea, whereas 4% were pathogens identified as Burkholderia glumae, Burkholderia gladioli and Acidovorax avenae ssp. avenae. Conclusions: This study reveals a broad morphological and genetic diversity of bacteria present on seed of a single rice cultivar. Significance and Impact of the Study: This line of work contributes to a better understanding of the microbial diversity present on rice seed and stresses its importance as a carrier of antagonists and pathogens.

Verticillium wilt is an increasing problem in European cauliflower production. In this study, several crop residues were screened for their ability to reduce the viability of microsclerotia when incorporated into soil. In addition, the role of fungitoxic volatiles and lignin in the crop residue-mediated reduction in microsclerotia viability was studied. Broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis), Indian mustard (Brassica juncea), ryegrass (Lolium perenne) and corn (Zea mays) were incorporated in naturally infested soil samples collected from two cauliflower fields in Belgium, labelled Is1 and S3. The effectiveness in reducing the viability of microsclerotia depended on the soil sample and on the type of residue. In the Is1 soil, broccoli, cauliflower and ryegrass incorporation significantly reduced the inoculum level by more than 90%, while Indian mustard significantly reduced numbers of viable microsclerotia by 50%. In the S3 soil, broccoli, cauliflower and Indian mustard were not effective, whereas ryegrass and corn incorporation reduced the microsclerotia level by 50% or more. In conclusion, incorporation of ryegrass and corn was more effective than incorporation of crucifer residues. In the conditions tested, fungicidal volatile compounds did not play an important role in Verticillium microsclerotia reduction in soil. Volatiles from broccoli and cauliflower did not affect microsclerotia viability in an in vitro bioassay, whereas the volatiles from Indian mustard killed the microsclerotia. Indian mustard incorporation in soil, however, only had a minor effect on microsclerotia viability. In the S3 soil, 1% (w/w) Kraft pine lignin, a waste product of the paper industry, had to be added to observe a significant reduction on the viability of microsclerotia, whereas in the Is1 soil, a significant effect was observed when 0.1% (w/w) Kraft pine lignin was added. Acid-insoluble lignin was extracted from all crop residues previously tested. Crop residues with high lignin content seemed to be more effective than crop residues with low lignin content. The reduction of Verticillium microsclerotia viability depended on lignin type and on crop structure, since lignin extracted from cauliflower leaves was more effective than lignin extracted from cauliflower stems and corn leaves were more effective than corn roots. Microsclerotia reduction was higher after fresh residue incorporation than after incorporation of their extracted acid-insoluble lignin, indicating that the effect of crop residue incorporation on microsclerotia viability cannot be explained solely by the effects of lignin. Incorporation of lignin-rich substrates in soil may open up new perspectives for integrated control of Verticillium.

The response of 13 European cauliflower cultivars to Verticillium longisporum was evaluated using two greenhouse tests and one in vitro inoculation test. The greenhouse tests involved dipping roots of 3-week-old seedlings in a conidial suspension or inoculating the soil of 3-week-old seedlings with Verticillium microsclerotia. The in vitro test involved the inoculation of 9-day-old seedlings with Verticillium conidia. Useful disease parameters were the area under disease progress curve and plant growth reduction for the greenhouse tests and fresh weight reduction for the in vitro test. Significant correlations were found among the three inoculation methods. Irrespective of the inoculation method used, cultivar Sernio was most resistant to V. longisporum, while Minaret was the most susceptible cultivar. The pathogen could be re-isolated from the hypocotyls and from the stem of Minaret 4 and 49 days after inoculation respectively, whereas V. longisporum could never be re-isolated from Sernio. These results suggest that the more resistant cauliflower cultivar Sernio can suppress the ascent and the proliferation of V. longisporum into the plant.

In April 2010, pink-orange spore masses that later turned brown were observed on 7 to 50% of the transplant lots during a routine screening of Belgian strawberry (Fragaria × ananassa, cv. Elsanta) for the latent presence of Colletotrichum acutatum using the petiole freeze method (4). These spore masses contained hyaline, canoe-shaped to allantoid conidia (mean size 7.5 × 1.8 μm), which is not consistent with C. acutatum spore morphology. Subsequently, a spore mass was transferred onto potato dextrose agar (PDA) and a gray-to-brown colony with whitish, aerial mycelium was produced, which is also not consistent with C. acutatum isolates. To identify the fungus, the ITS1-5.8S-ITS2 rDNA region was amplified by PCR and sequenced. The 485-bp region was 100% identical to that of Pilidium concavum specimen voucher BPI 1107275 (GenBank Accession No. AY487094). P. concavum (Desm.) Höhn. (synanamorph Hainesia lythri; teleomorph Discohainesia oenotherae) is a pathogen of strawberry causing tan-brown rot of fruit and is a common secondary invader of roots and dead strawberry plant parts (3). A recent strain of P. concavum from strawberry, isolate UPL 50, obtained from Brazil (L. Zambolim, Univ. Fed. de Viçosa, personal communication) showed similar colony, microscopic (mean spore size of 6.8 × 1.8 μm), and molecular (ITS sequence 98% identical to that of P. concavum specimen voucher BPI 1107275) features as the Belgian isolate. Pathogenicity tests were conducted on mature strawberry fruits by submerging 15 fruits per isolate for 3 min in a conidial suspension (2 × 106 conidia ml–1 of water) obtained from a 2-week-old colony on PDA. Controls were submerged in sterile distilled water. The inoculated fruits were incubated in a moist chamber at 25°C. Sunken, yellowish brown lesions with pink and later orange-brown spore masses were observed starting 3 days after inoculation on 88 and 94% of the fruit for the Brazilian and Belgian isolate, respectively. The control fruits remained healthy. The fungal isolates were reisolated from symptomatic fruits and their identity was confirmed based on morphological features. During a strawberry field survey in July 2010 in Sint-Truiden (Belgium), lesions typical of those described above were observed on eight strawberry fruits (cv. Elsanta). The fungus was isolated from the symptomatic tissue of two fruits and characterized as described above. Since P. concavum was latently present on strawberry transplants and caused disease on the fruits in the field, we conclude that P. concavum is a potential threat for Belgian strawberry production. Moreover, no strawberry cultivars with resistance to the pathogen have been reported. The disease has previously been reported on strawberry in South America and Poland (1,2), but to our knowledge, this is the first report of P. concavum on strawberry in Belgium. Although the spore and colony morphology of P. concavum is different from C. acutatum, the spore masses of P. concavum can easily be confused with the spore masses of C. acutatum when using the freeze method. This suggests the need for microscopic analysis of these spore masses during routine analyses. References: (1) L. Cedeno et al. Interciencia 26:113, 2001. (2) U. P. Lopes et al. New Dis. Rep. 21:7, 2010. (3) J. L. Maas. Compendium of Strawberry Diseases. The American Phytopathological Society St. Paul, MN, 1998. (4) J. C. Mertely and D. E. Legard. Plant Dis. 88:407, 2004.