Search

Spinach growers face increasing problems of damping-off in the production of fresh-market (baby-leaf) spinach due to increasing restrictions on chemical treatments. Damping-off-tolerant cultivars are increasingly important, requiring proper evaluation methods. From three locations in The Netherlands with a history of spinach cultivation and from one location in France, potential damping-off pathogens were isolated from the soil, identified to species or genus, and tested for their pathogenicity. Pythium ultimum was the most prevalent pathogen in those fields, causing spinach pre- and postemergence damping-off. Eight spinach cultivars with two or three seed lots each were evaluated at the same field locations and in a greenhouse with soil sampled from one of the Dutch field sites. Preemergence damping-off was more discriminating for differences among the cultivars than postemergence damping-off. Variation in levels of infection among trials, replicates, and seed lots of the same cultivars emphasized the need for a more standardized phenotyping assay. For such an assay, a cornmeal-sand-based inoculum with a pathogenic P. ultimum isolate was added to a substrate mixture of sand, perlite, and vermiculite and moistened until 50% water-holding capacity, in which spinach seeds were incubated for 10 days in a dark climate cabinet at 15°C, including a control treatment without P. ultimum inoculum. The assay showed reproducible results for discriminating differences in preemergence damping-off tolerance levels among seed lots. However, cultivar differences in preemergence damping-off tolerance levels could not be confirmed due to a large variation among seed lots that needs further investigation.

Intensive agricultural crop production can lead to a decline in biological soil characteristics and functions, such as soil microbial biomass and activity, carbon and nutrient cycling and soil suppressiveness, important for the sustainable production of food and feed. There is a need to understand how those soil functions can be improved by agricultural practices. In a long-term field study, we assessed whether reduced tillage could enhance soil biological parameters and soil suppressiveness against the plant-pathogenic fungus Rhizoctonia solani AG2-2IIIB and bacterium Streptomyces scabies. Soil suppressiveness was assessed in bioassays with a susceptible crop while adding pathogen inoculum. Undisturbed soil cores (0–12 cm) were used in these bioassays to include the soil structural aspects, which were likely affected by the tillage treatments. Reduced i.e., non-inversion, tillage was compared with conventional ploughing treatment over 6 years. We found that reduced tillage led to an increase in bacterial and fungal biomass, labile carbon and nitrogen and an increase in the abundance of potential bacterial antagonists, compared to conventional tillage in the upper 12 cm. However, the increase of these microbial parameters did not lead to consistent changes in soil suppressiveness against both R. solani and S. scabies in response to the tillage treatment. Rather, disease suppressiveness varied significantly between field and year of sampling but was not correlated to any of the assessed soil parameters. Thus while reduced tillage can be beneficial for soil biology, other measures will have to be investigated for inducing R. solani and S. scabies disease suppressiveness.

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.

Apart from improving the physical and chemical condition of arable soils, cover crops have the potential to boost and activate selected soil microbiota that could contribute to improved nutrient cycling and strengthened disease suppressiveness. However, a main crop can only benefit from cover crop-induced microbial shifts if these persist until the onset of the main growing season. Here, we map the persistence of microbiome changes by cover crops over time. We performed a field experiment on a sandy soil with ten different cover crop monocultures belonging to five plant families, one cover crop mixture and a fallow control. Cover crops were grown for 4.5 months under field conditions in 70-L bottomless containers in a random block design with eight replications. We studied the total (DNA-based) and the potentially active (RNA-based) microbial fractions at the onset of the main growing season, and just after the harvest of the main crop, potato (respectively 3.5 and 10 months after cover crop termination), through MiSeq sequencing. All cover crops tested induced shifts in the soil microbiome that lasted at least until the onset of the main growing season. Cover crop treatments gave rise to species and even cultivar-specific microbial footprints, and - although roughly the same trends were observed - DNA-based microbial shifts were not necessarily paralleled by similar changes at RNA level. We conclude that cover crops have the potential to act as handles to steer the soil microbiome in a way that is supportive of sustainable crop production.

Cover crops are used in agriculture to minimise soil erosion, prevent nutrient leaching and increase soil organic matter content. Cover crops can also be grown to stimulate the soil microbial community to improve soil biological conditions. Despite their widespread use, little is known about the impact of different cover crop species on the composition and activity of the soil microbiome. Here we investigate the effect of distinct cover crop species on the rhizosphere microbiome and to characterise both the resident (DNA-based) and the active (RNA-based) fractions of the bacterial, fungal, protist and metazoan communities in the cover crops rhizosphere. We conducted a field experiment using 70-litre bottomless containers in which we grew ten monocultures of commonly used cover crop species belonging to five plant families, and an unplanted control treatment (fallow). The total DNA and RNA were extracted from soil and the bacterial, fungal, protistan and metazoan communities were characterized using Illumina MiSeq sequencing. We found that all cover crop species significantly impacted the resident and active microbial community composition. Moreover, cover crops showed distinct selection strengths on their rhizospheres. Together with borage (Boraginaceae), oilseed radish (Brassicaceae) was shown to provoke the strongest microbial shifts, in part attributable to a promotion of the bacterial family Pseudomonadaceae and a repression of Microascaceae in the rhizosphere. Lentil (Fabaceae) showed enrichment in fungal taxa, including Trichocomaceae and fungal members of the Glomerales order, whereas black oat, hybrid ryegrass (both Poaceae) and marigold (Asteraceae) induced relatively mild changes in the soil microbial communities. Predominantly, differences in selection strengths were consistent among the four organismal groups under investigation and were observed in both the active and resident communities. Our work provides a broad baseline for the effects of cover crops on four organismal groups, which may facilitate future cover crop selection to advance soil health.

Seed health is an indispensable prerequisite of food security. While the toolkit of plant protection products is currently limited, evidence suggests that the seed microbiome could protect seeds from pathogens. Thus, given their possible disease suppressive potential, we tested 11 different pathosystems to achieve the following proof-of-concept: seed microbiomes can be beneficial for seed health through conferring disease suppression. This study focused on beetroot, onion, spinach, pepper, coriander, red fescue and perennial ryegrass seeds, with each crop being challenged with one or two from a total of six pathogens, namely Pythium ultimum (or a Pythium sp.), Setophoma terrestris, Fusarium oxysporum, Phytophthora capsici, Laetisaria fuciformis and a mix of Puccinia sp. isolates. Each seed lot of each crop was tested with and without treatment with a disinfectant as a proxy for comparing intact seed microbiomes with seed microbiomes after partial elimination by disinfection. We found disease suppression in two pathosystems. Beetroot and spinach seed lots were able to suppress disease caused by P. ultimum when their microbiomes were intact but not after seed disinfection. We speculate that this relates to the microorganisms residing on and in the seed. Yet, seed microbiome disease suppression was not found in all pathosystems, highlighting the variation in seed morphology, plant cultivars, pathogens and seed disinfection treatments. A holistic understanding of the characteristics of seeds that harbour suppressive microbiomes as well as the pathogens that are sensitive to suppression could lead to more targeted and informed seed processing and treatment and, consequently, to the sustainable management of seedling diseases.

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., 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.

Approximately 100 years after the first introduction of Dutch elm disease (DED) in Europe, an effective commercial biocontrol product is available to protect susceptible elm trees against DED transmission by beetles. Injection of trees with conidiospores of the fungus Verticilium albo-atrum isolate WCS850, product name DutchTrig®, reduces infection to less than 0.2% of treated trees. This biocontrol agent, its mode of action, application, efficacy and limitations in controlling DED are described in this chapter.

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.

Worldwide there is an enormous interest in microbial indicators for soil quality, since this reflects the potential capacity for soil ecosystem functions i.e. nutrient cycles, carbon storage, biodiversity and resilience to climate change. Farmers are anxious to measure the effects of different soil management practices in order to improve soil quality and attain sustainable food production. Despite the rapid developments in (molecular) measurement techniques, adequately validated and affordable methods for field measurements on soil microbial activity are still lacking. Nowadays, farmers participate in campaigns to bury cotton undies in order to measure biological activity in their fields (Soil your undies). If there’s not much left of the undies after a couple of months, this supposedly indicates good soil health. Of course this is by no means a quantitative nor validated indicator.An elegant, cheap and simple method to measure biological activity in soil is the Tea Bag Index (TBI). This method was developed to determine the global variation in decomposition rate of organic matter by the soil microflora as influenced by abiotic circumstances. The TBI consists of two parameters describing decomposition and stabilization of organic matter by measuring weight loss of green tea and rooibos tea bags that have been buried in the soil for three months. The method is designed to discriminate contrasting ecosystems and, within ecosystems, differences in factors such as soil temperature and moisture content (Keuskamp et al. 2013, doi: 10.1111/2041-210X.12097).Our research aimed to assess the possibility to use the TBI as an indicator for soil microbial activity, considering its sensitivity and robustness to discriminate between agricultural soil management practices that are known to have a significant impact on soil microbial diversity and activity. The responsiveness to soil pasteurization and organic amendments was investigated under both controlled and field conditions. The TBI decomposition rate differed significantly between both tea varieties (green tea > rooibos tea). Organic amendments had little or no effect. The TBI-results were plotted against some more established biochemical indicators which are sensitive to soil management and often related to microbial biomass, i.e. hot water extractable carbon, potentially mineralizable nitrogen and fungal biomass. Results are discussed, as well as factors which complicate the interpretation of TBI data with respect to soil microbial activity.

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.

Tien zeer uiteenlopende organische producten zijn onderzocht op hun vermogen om ziektewering van een bodem te verhogen. In potproeven werd aangetoond dat een aantal van deze producten de ziektewering van zandgrond tegen Rhizoctonia solani en Meloidogyne hapla konden stimuleren. Vervolgens zijn de organische producten onder praktijkomstandigheden in de teelt van aardappel en suikerbiet getoetst. Bij een dosering van de producten conform de bemestingsadviezen waren de opbrengsten van de gewassen vergelijkbaar met een kunstmest bemeste controle en werd afhankelijk van de samenstelling van de producten tot 160, 50 en 200 kg/ha aan N, P en K bemesting bespaard (bij aardappel). De producten hadden onder veldomstandigheden een geringe invloed op de ziektewerende eigenschappen van de grond: Pythium ziektewering was in enkele gevallen verhoogd, maar er was geen meetbare verhoging van ziektewering tegen Rhizoctonia en Meloidogyne. Daarnaast zijn diverse (biologische) bodemparameters bepaald.

This report describes two bioassays for testing disease suppression by BSF frass in an agricultural soil with two crops: sugar beet (Beta vulgaris) and cress (Lepidium sativum). In the bioassay with beet, soil was infected with the plant-pathogenic fungus Rhizoctonia solani while in the bioassay with cress, soil was non-infected or infected with the plant-pathogenic fungus Pythium ultimum. Both concentrations of the BSF frass (7.5 and 15 g/kg soil) proved to be useful as soil application, leading to good plant emergence and growth of sugar beet and cress plants. The highest concentration of BSF frass (15 g/kg soil, both as total product) had a large fertilizer effect. In experiments with cress without addition of Pythium (i.e. with only natural soil infections), disease suppression with BSF frass was found. In all tests, keratin was added as a positive control treatment, which had a higher disease suppression effect than BSF frass but comparable fertilizer effects.

To create more resilient growing systems for greenhouse crops, the efficacy of different organic amendments alone and in combination with the biocontrol strain Pseudomonas chlororaphis 4.4.1 was tested to suppress Pythium disease in tomato plants. Four independent greenhouse experiments were performed with young tomato plants in potting soil. Inoculating the pathogen Pythium aphanidermatum to the potting soil prior to sowing resulted in significant losses of tomato plants; i.e. 94–98% healthy plants in pathogen-free control compared to 43–68% healthy plants when the pathogen was added. P. chlororaphis 4.4.1 inoculations increased the number of healthy plants in the potting soil up to 80% on average; soil and seed treatment were both effective. Numbers of P. chlororaphis in the rhizosphere had increased 4 to 100 fold 3 weeks after its inoculation (qPCR detection). All compost types reduced Pythium disease in potting soil resulting in 80–95% healthy plants. Animal bone char was not effective against P. aphanidermatum, whereas with plant-based biochar there was an effect, although not significantly different from the control treatment. Phosphorous and potassium uptake by the plants were elevated by the different organic amendments. These results demonstrate the potential of the organic amendments to enhance sustainability of growing media such as potting soil by increasing its disease suppressiveness, the re-use of nutrients and replacement of peat by using organic amendments. In addition, inoculating the growing medium or tomato seeds with the biocontrol strain P. chlororaphis 4.4.1 enhanced Pythium control in the susceptible growing media.

Soil suppressiveness to pathogens is defined as the capacity of soil to regulate soil-borne pathogens. It can be managed by agricultural practices, but the effects reported so far remain inconsistent. Soil suppressiveness is difficult to predict and for this reason different soil properties have been linked to it with the aim to find informative indicators, but these relationships are not conclusive. The objectives of this study were i) to test if soil suppressiveness is affected by long-term agricultural management such as tillage and organic matter (OM) addition; ii) to understand the direct and indirect relationships between soil suppressiveness and labile organic carbon fractions; and iii) to understand the relationship between soil suppressiveness and other chemical, physical and biological soil quality indicators. We measured soil suppressiveness with a bioassay using Pythium ultimum - Lepidium sativum (cress) as a model system. The bioassay was performed in soils from 10 European long-term field experiments (LTEs) which had as main soil management practices tillage and/or organic matter addition. We found that the site had a stronger influence on soil suppressiveness than agricultural practices. Reduced tillage had a positive effect on the suppressive capacity of the soil across sites using an overall model. Organic farming and mineral fertilization increased soil suppressiveness in some LTEs, but no overall effect of OM was found when aggregating the LTEs. Soil suppressiveness across LTEs was linked mainly to microbial biomass and labile carbon in the soil, but not to total soil organic matter content. From structural equation modelling (SEM) we conclude that labile carbon is important for the maintenance of an abundant and active soil microbial community, which is essential for the expression of soil suppressiveness. However, soil suppressiveness could only partly (25%) be explained by the soil parameters measured, suggesting that other mechanisms contribute to soil suppressiveness such as the presence and the activity of specific bacterial and fungal taxa with high biocontrol activity.

The authors regret < To the editor of Soil Biology and Biochemistry, While reviewing the data for another manuscript I found an error in the calculation of the decomposition measured by the tea bag method that was included in the article: “Soil suppressiveness to Pythium ultimum in ten European long-term field experiments and its relation with soil parameters” by Bongiorno G., Postma J., Bünemann E., Brussaard L., de Goede R., Mäder P., Tamm L., and Thürig B. published in 2019 in Soil Biology and Biochemistry 133: 174–187. The error made was this: to calculate the tea bag decomposition, the final weight of the tea bag instead of the % mass loss (i.e. 1-final weight/initial weight) was taken. Hence, the negative partial correlation originally found should be positive, which actually makes more sense. I am very sorry for this unfortunate error and herewith provide you with the corrected results, see the correct rows in the tables at the end of this message. Below, the consequences for the conclusions of the current manuscript are described. In summary: the error found in the final calculation of tea bag decomposition does not undermine the messages and results of the article. In detail: Text: In the text below, which can be found in the beginning of the result section 3.3, I changed the incorrect text: “Bivariate correlation analysis showed that soil suppressiveness (SSni) (calculated from the management treatment samples) was positively associated with higher values of various chemical (pH, total N, cation exchange capacity (CEC), Ca and K), physical (water holding capacity (WHC), silt, clay, penetration resistance), microbial parameters (microbial biomass C and N (MBC and MBN)), soil respiration (SR), microbial quotient (qMic), earthworm number and biomass, and labile carbon fractions (hydrophilic dissolved organic carbon (Hy-DOC), permanganate oxidizable carbon (POXC) and hot water extractable carbon (HWEC)) (Table S4). In contrast, we found negative correlations with C to N ratio (C/N), bulk density (BD), sand, tea bag decomposition, dissolved organic carbon and hydrophilic organic carbon specific ultraviolet absorbance (DOC SUVA and Hy SUVA). The partial correlation showed that after normalization for structural differences between the LTEs (i.e. for the pedoclimatic characteristics) higher values of total N, MBC, soil respiration, qMic, earthworm number, tea bag decomposition, Hy SUVA, POXC, HWEC and carbon in the particulate organic matter (POMC) were associated with higher values of SSni, while higher values of C to N ratio, and DOC SUVA were associated with lower values of SSni (Table 4).“ Table 1 (see below): The unit of tea bag decomposition is % mass loss and not g mass loss. Tables 4 and 5 (see below): The now positive partial correlation of tea bag decomposition with soil suppressiveness is similar to that of MBC, soil respiration and qMic, which makes more sense. However, decomposition based on the tea bag method is not one of the most important variables in later analyses (simple mixed linear models in Table 5), so it would not have been used in the structural equation model and it is not at variance with later results. Table S4 (see below): The now negative bivariate correlation of the bag decomposition with soil suppressiveness is due to the fact that the long-term field experiments CH1 and CH2 have high values of soil suppressiveness but low decomposition values. I, therefore, would like to request a correction of the manuscript in the indicated section, and I submit this request together with the corrections in the contact form available on the Elsevier Journal Article Publishing Support Center. Sincerely, Giulia Bongiorno [Table presented] [Table presented] [Table presented] [Table presented] The authors would like to apologise for any inconvenience caused.

Since 1992 elm trees have been treated with a biological control product Dutch Trig® to protect them from infection by Ophiostoma novo-ulmi causing Dutch elm disease (DED). The active ingredient of the biocontrol product consists of the fungus Verticillium albo-atrum strain WCS850. A conidiospore suspension of this fungus is injected into the vascular system of elm trees at a height of 1.3 m. This biocontrol product prevents healthy elm trees from fungal infection transmitted by elm bark beetles. Dutch Trig®, however, does not protect already infected trees or trees connected with diseased trees via root grafts. Since 2010, only 0.1 % of the injected elms became infected with DED through beetle transmission and an additional 0.4 % of the treated elms were infected through root contact in the Netherlands. Regression analysis considering all injected elm trees in the Netherlands since 1992 indicated that DED infection through beetle transmission had significantly decreased during the 24 years application of Dutch Trig®. In 2015, approximately 28,300 trees in five countries (Netherlands, USA, Germany, Canada and Sweden) were treated with Dutch Trig® to protect valuable susceptible elm trees, mainly in urban environments.

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. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0310-6) contains supplementary material, which is available to authorized users.

Four taxonomically different bacteria, with the ability to mobilize phosphate (P) and to colonize animal bone charcoal (ABC), were tested for their capacity to control plant pathogens. Tests were performed in the greenhouse with young tomato plants in (potting) soil and in rockwool. Plants were infested with Pythium aphanidermatum and Fusarium oxysporum f.sp. radicis-lycopersici (FORL) causing respectively damping off and crown and root rot. ABC is a porous, phosphorous containing waste product from the food industry, and was used as carrier to introduce the bacteria into the growing media. Scanning electron microscopy (SEM) pictures showed the intensive colonization of the bacteria in the interior of ABC. Of the four tested strains, Pseudomonas chlororaphis 4.4.1 was most effective in controlling the diseases. It controlled P. aphanidermatum and FORL in tomato in each of the tests. The strain appeared to be a very good root colonizer, since 1–8% of the cultural bacterial population on the tomato roots or in rhizosphere soil consisted of the introduced strain. Population densities of P. chlororaphis 4.4.1 were 0.5–5 × 107 CFU g-1 root or rhizosphere soil. Peanibacillus polymyxa 12.4.1 and Streptomyces pseudovenezuelae 13.4.2 significantly controlled P. aphanidermatum in two tests in potting soil, whereas Bacillus pumilus 4.4.2 was not effective. FORL could be controlled by B. pumilus 4.4.2 and S. pseudovenezuelae 13.4.2 in only part of the tests, whereas P. polymyxa 12.4.1 was not effective. ABC is a novel carrier for delivery of biocontrol bacteria into soil or substrate and combines biocontrol with recycling a phosphorous-rich waste product

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2191-z) contains supplementary material, which is available to authorized users.

Disease suppressiveness against Rhizoctonia solani AG 2-1 in cauliflower was studied in two marine clay soils with a sandy loam texture. The soils had a different cropping history. One soil had a long-term (40 years) cauliflower history and was suppressive, the other soil was conducive and came from a pear orchard not having a cauliflower crop for at least 40 years. These two soils were subjected to five successive cropping cycles with cauliflower or remaining fallow in a greenhouse experiment. Soils were inoculated with R. solani AG 2-1 only once or before every crop. Disease decline occurred in all treatments cropped with cauliflower, either because of a decreased pathogen population or increased suppressiveness of the soil. Disease suppressiveness tests indicated that the conducive soil became suppressive after five subsequent cauliflower crops inoculated each cycle with R. solani AG 2-1. Suppressiveness of all treatments was measured in a seed germination test (pre-emergence damping-off) as well as by measuring the spread of R. solani symptoms in young plants (post-emergence damping-off). Results showed that suppressiveness was significantly stimulated by the successive R. solani inoculations; presence of the cauliflower crop had less effect. Suppressiveness was of biological origin, since it disappeared after sterilization of the soil. Moreover, suppressiveness could be translocated by adding 10% suppressive soil into sterilized soil. The suppressive soil contained higher numbers of culturable filamentous actinomycetes than the conducive soil, but treatments enhancing suppressiveness did not show an increased actinomycetes population. The suppressiveness of the soil samples did also not correlate with the number of pseudomonads. Moreover, no correlation was found with the presence of different mycoparasitic fungi, i.e. Volutella spp., Gliocladium roseum, Verticillium biguttatum and Trichoderma spp. The suppressive soil contained a high percentage of bacteria with a strong in vitro inhibition of R. solani. These bacteria were identified as Lysobacter (56%), Streptomyces (23%) and Pseudomonas (21%) spp. A potential role of Lysobacter in soil suppressiveness was confirmed by quantitative PCR detection (TaqMan), since a larger Lysobacter population was present in suppressive cauliflower soil than in conducive pear orchard soil. Our experiments showed that successive cauliflower plantings can cause a decline of the damage caused by R. solani AG 2-1, and that natural disease suppressiveness was most pronounced after subsequent inoculations with the pathogen. The mode of action of the decline is not yet understood, but antagonistic Lysobacter spp. are potential key organisms.

Aims: To assess the interactions between Coniochaeta ligniaria F/TGF15 obtained from torrefied grass fibers (TGF) and selected bacteria from the same substrate. Methods and Results: Upon coinoculation on potato dextrose agar, Pseudomonas putida 15/TGE5, Methylobacterium radiotolerans 56/TGF10, Serratia plymutica 23/TGE5, Pseudomonas corrugata 31/TGE5, Leifsonia xyli subsp. xyli 66/TGF10, Mycobacterium anthracenicum 70/TGF15 and Agromyces aurantiacus 95/TGF15 were translocated by C. ligniaria, but not in the absence of the fungus. Pseudomonas putida, P. corrugata, L. xyli subsp. xyli and A. aurantiacus were able to grow on compounds released by the fungus, but not M. radiotolerans. Antagonism towards C. ligniaria was observed for S. plymutica and P. corrugata. Pseudomonas putida was translocated by the fungus in TGF up to at least 45 mm. It also multiplied on the hyphae of C. ligniaria in TGF, reaching CFU densities of log 8·4 g−1 dry TGF in 20 d, while the strain could not grow in nonfungal TGF. Methylobacterium radiotolerans was not translocated by the fungus in TGF. Conclusions: Several of the selected bacteria could grow on the compounds released by the fungus, whereas two bacteria inhibited or killed the fungus. Significance and Impact of the Study: It is shown that C. ligniaria has a dual role in bacterial colonization of TGF, being crucial for the detoxification of TGF, meanwhile stimulating growth and translocation of a consortium of plant-growth-promoting bacteria.

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.

This study investigates how thermally treated (i.e., torrefied) grass, a new prospective ingredient of potting soils, is colonized by microorganisms. Torrefied grass fibers (TGF) represent a specific colonizable niche, which is potentially useful to establish a beneficial microbial community that improves plant growth. TGF and torrefied grass extracts (TGE) were inoculated with a suspension of microorganisms obtained from soil. Sequential microbial enrichment steps were then performed in both substrates. The microbial communities developing in the substrates were assessed using cultivation-based and cultivation-independent approaches. Thus, bacterial isolates were obtained, and polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) analyses for bacterial communities were performed. Partial sequencing of the 16S ribosomal RNA gene from isolates and bands from DGGE gels showed diverse communities after enrichment in TGE and TGF. Bacterial isolates affiliated with representatives of the alpha-proteobacteria (Methylobacterium radiotolerans, Rhizobium radiobacter), gamma-proteobacteria (Serratia plymuthica, Pseudomonas putida), Cytophaga-Flavobacterium-Bacteroides (CFB) group (Flavobacterium denitrificans), beta-proteobacteria (Ralstonia campinensis), actinobacteria (Cellulomonas parahominis, Leifsonia poae, L. xyli subsp. xyli, and Mycobacterium anthracenicum), and the firmicutes (Bacillus megaterium) were found. In TGE, gamma-proteobacteria were dominant (61.5% of the culturable community), and 20% belonged to the CFB group, whereas actinobacteria (67.4%) and alpha-proteobacteria (21.7%) were prevalent in TGF. A germination assay with lettuce seeds showed that the phytotoxicity of TGF and TGE decreased due to the microbial enrichment.

The efficacy of different organic soil amendments on disease suppression to Rhizoctoniasolani AG 2-2IIIB was tested in a bio-assay with sugar beet as a test plant. Lysobacter populations in soil were quantified as a possible mechanism for disease suppression. Disease spread through the bio-assay tank was significantly reduced up to 86, 83, 52, and 48% after amending the non-sterilized soil with yeast or chitin at a rate of 0.3% (w/w) in consecutive experiments. Inexpensive protein-rich waste products from food industry (i.e., feather, hoof, meat, blood and fish meal) also effectively increased Rhizoctonia-disease suppression. Several plant-derived products (e.g., spent mushroom compost, dried algae, spent brewer’s grain, Brassica seed meal) were not effective. Lysobacter populations naturally present in the soil were increased 3–10 fold (measured by a TaqMan quantitative PCR) in soils amended with organic compounds that stimulated Rhizoctonia-disease suppression. The role of Lysobacter as a key factor in Rhizoctonia-disease suppression, however, could not be confirmed by adding Lysobacter isolates to a sterilized soil amended with yeast or chitin. Hence, we hypothesize that unexplored biological factors were involved in disease suppression, since the tested soil became conducive after gamma-sterilization. The consistent enhancement of Rhizoctonia-disease suppression in sugar beet with yeast and chitin amendments, and the efficacy of inexpensive protein-rich waste products such as feather meal and hoof meal in our bio-assays, warrants further study in field experiments.

The extent of soil microbial diversity is seen to be critical to the maintenance of soil health and quality. Different agricultural practices are able to affect soil microbial diversity and thus the level of suppressiveness of plant diseases. In a 4-year field experiment, we investigated the microbial diversity of soil under different agricultural regimes. We studied permanent grassland, grassland turned into arable land, long-term arable land and arable land turned into grassland. The diversity of microbial communities was described by using cultivation-based and cultivation-independent methods. Both types of methods revealed differences in the diversities of soil microbial communities between different treatments. The treatments with higher above-ground biodiversity generally maintained higher levels of microbial diversity. Moreover, a positive correlation between suppression of Rhizoctonia solani AG3 and microbial diversity was observed. Permanent (species-rich) grassland and grassland turned into maize stimulated higher microbial diversities and higher levels of suppressiveness of R. solani AG3 compared with the long-term arable land. Effects of agricultural practices on Bacillus and Pseudomonas communities were also observed and clear correlations between the levels of suppressiveness and the diversities of these bacterial groups were found. This study highlighted the importance of agricultural management regime for soil microbial community structure and diversity as well as the level of soil suppressiveness.

Postma, J., Geraats, B. P. J., Pastoor, R., and van Elsas, J. D. 2005. Characterization of the microbial community involved in the suppression of Pythium aphanidermatum in cucumber grown on rockwool. Phytopathology 95:808-818. 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.

Lysobacter enzymogenes strain 3.1T8 is a potential biocontrol agent of Pythium aphanidermatum (Edson) Fitzp. in cucumber (Cucumis sativa L.). It showed in vitro antagonistic activity, hemolytic activity, and the production of a surface active compound, which decreased in media of increasing strength. One or more low molecular compounds, present in culture filtrates of 3.1T8, caused rapid immobilization of zoospores of P. aphanidermatum and inhibited cyst germination. Bioassays for suppression of root and crown rot were performed with 2-week-old cucumber plants in nutrient solution. Complete disease suppression occurred after addition of a suspension of unwashed cells grown on nutrient-poor R2A, or the filtrate of this suspension, 1 day before inoculation with zoospores. Application of washed cells caused slight suppression in one of two bioassays only. No disease suppression occurred when bacterial inoculum grown on 1/10 TSBA or in 1/10 TSB was used. In two bioassays on cucumber plants grown for 4–5 weeks in 18-L containers, application of R2A-grown cells of 3.1T8 did not cause disease suppression. Density of 3.1T8, monitored using immunofluorescence colony staining, decreased at least 100-fold during these experiments to approximately log 3–4 cfu ml-1 of nutrient solution and log 5–6 cfu g-1 roots. Overall, the findings indicated a strong influence of nutritional resources on production of antifungal compounds by strain 3.1T8. The lack of disease suppression in hydroculture containers suggested that in situ production of the compounds was insufficient or absent, although the bacteria persisted in the nutrient solution and on the roots of cucumber. [KEYWORDS: Lysobacter enzymogenes ; Pythium aphanidermatum ; Cucumis sativa L. ; Hydroponics ; Rockwool ; Biosurfactant ; Antibiotic ; Root colonization ; Quorum sensing]

In this study results are presented of investigations on the development of a cucumber crop in a closed rockwool system and on the potential change in microbial suppressiveness due to different disinfection methods after inoculating part of the plants with the fungal pathogen Pythium aphanidermatum. Two trials were executed at which the core treatments were disinfection (UV, slow filtration and no disinfection as control), while additional treatments were priming half of the systems before planting either with an 'old' nutrient solution from a former cucumber crop (spring trial) or an actinomycetes suspension (autumn trial). Crop developments (yield, disease and root development) and composition of the microflora were recorded. Non-inoculated plants showed that disinfection is needed either with UV or slow (sand) filtration to avoid spread of the pathogen; yield and root development was better and disease symptoms were less compared to the control. The hypothesis that passive disinfection might realise a more suppressive environment against Pythium aphanidermatum could not be proved, since inoculated plants showed a similar yield, disease and root development after the different disinfection treatments. Priming the nutrient solution with an 'old' solution did not show significant differences in the composition of the microflora either by plate-counts or by PCR-DGGE. However, the addition of actinomycetes before planting resulted a 100 to 1000 fold increase of the actinomycetes population in the cropping system up to the end of the trial. In the spring trial, differences within the microflora as a result of the disinfection treatments were only detected for pseudomonads. In the autumn trial, differences in numbers of pseudomonads, actinomycetes and fungi were significant in the treated nutrient solution, but not in the drain. With PCR-DGGE, differences in the bacterial profile were detected in autumn in both the drain and treated solution; i.e. less bands (corresponding to certain bacterial species) occurred in the UV treatment compared to slow filtration and control

Compost amended soil has been found to be suppressive against plant diseases in various cropping systems. The level and reproducibility of disease suppressive properties of compost might be increased by the addition of antagonists. In the present study, the establishment and suppressive activity of two fungal antagonists of soil-borne diseases was evaluated after their inoculation in potting soil and in compost produced from different types of organic waste and at different maturation stages. The fungal antagonists Verticillium biguttatum, a mycoparasite of Rhizoctonia solani, and a non-pathogenic isolate of Fusarium oxysporum antagonistic to fusarium wilt, survived at high levels (103–105 CFU g–1) after 3 months incubation at room temperature in green waste compost and in potting soil. Their populations faded out in the organic household waste compost, especially in the matured product. In bioassays with R. solani on sugar beet and potato, the disease suppressiveness of compost increased or was similar after enrichment with V. biguttatum. The largest effects, however, were present in potting soil, which was very conducive for the disease as well as the antagonist. Similar results were found in the bioassay with F. oxysporum in carnation where enrichment with the antagonistic F. oxysporum had a positive or neutral effect. We foresee great potential for the application of antagonists in agriculture and horticulture through enrichment of compost or potting soil with antagonists or other beneficial micro-organisms.

Enhancement of disease suppressive properties of soils will limit disease development, thus, being of great importance for sustainable agricultural farming systems. The current research demonstrated that suppressiveness against Rhizoctonia solani AG 2-2IIIB in sugar beet could be elevated in different soils. Feather meal was most effective and disease spread was reduced with 36-87% in clay as well as in sandy soils. Chitin was effective in clay, but less in sand. Whereas suppressiveness was hardly elevated in loess soils. The potential to enhance suppressiveness was lost after frost (4 weeks -15°C) as well as after flooding soil during 1 or 4 weeks. R. solani was not only suppressed in sugar beet seedlings, but also in cauliflower with AG 2-1 and lettuce with AG 1-1B. R. solani was not suppressed in lettuce with AG 4 HGI and carrot with AG 2-2IIIB. These results indicate that Rhizoctonia-suppressiveness can be enhanced in different soils and different crops, but specificity of the suppressiveness among Rhizoctonia-crop combinations is expected

Phytophthora cactorum is an important threat in strawberry propagation and production. No reliable non-chemical control measures are available. Therefore different control strategies were tested. Spread of pathogen infection can be reduced by disinfection of the irrigation water. Slow sand filtration effectively removed P. cactorum from drain water, i.e., disease severity in strawberry plants was reduced with 45 to 65% and rhododendron baits showed a 95-100% reduction of P. cactorum after the drain water had passed the sand filter. However, upstream movement of P. cactorum was detected within the unit. A complementary strategy to reduce the damage caused by P. cactorum is to enhance disease suppressive properties of the soilless substrate. Pasteurization of the substrate mixtures showed increased levels of disease, indicating that biological factors might play a role in disease suppression. Antagonistic bacteria inhibiting growth of P. cactorum were present in roots, rhizosphere as well as in the crown. However, compost amendment or other additions did not elevate suppressiveness. Further experiments were performed where different beneficial microorganisms and/or elicitors were added. The most promising treatments were salicylic acid, followed by inoculation with the endophytic fungus Acremonium strictum and growing Lepidium sativum previous to the crop

Bacterial communities from the rhizosphere of cucumber were characterized with respect to growth rates and carbon source utilization, in order to develop a selection strategy for biocontrol agents against Pythium aphanidermatum. Rhizosphere samples were collected from different root regions (root tips, the root base, and the intermediate region where lateral roots emerge) and developmental stages (the seedling, vegetative, and generative stage) from plants cultivated on reused rockwool. By colony counts on 1/10 strength TSA on subsequent days after plating, percentages of fast- and slow-growing isolates (i.e., forming visible colonies within 2 days, or after 3 or more days, respectively) were determined for each rhizosphere sample. At all plant developmental stages, root tips had the highest percentages of fast growing isolates, and root bases the lowest. During plant growth, the relative amounts of slowly growing bacteria increased. Community-level carbon source utilization was determined for the different rhizosphere samples with Biolog GN plates. Principal component analysis showed that rhizosphere samples from different developmental stages and root locations had distinct carbon source utilization patterns. Communities from root tips of seedlings showed the highest utilization of several monosaccharides. Communities from tips and intermediate regions of plants in the vegetative stage utilized relatively many amino acids and several organic acids, and in the generative stage, more di- and polysaccharides were used. Root base samples scored low with respect to carbon source utilization, except for some disaccharides. From the different rhizosphere samples, 826 bacteria, randomly collected from 1/10 strength TSA plates, were screened on the utilization of 9 carbon sources. The 9 selected carbon sources were chosen because they are reported to occur in the rhizosphere, to be used by the zoospores of Pythium in the infection process, or appeared to be discriminant in the analysis of community-level carbon source utilization performed in this study. It appeared that monosaccharides (glucose and fucose), amino acids (alanine and asparagine), and organic acids (galacturonic, succinic, and linoleic acid) were used for growth mainly by bacteria from the root tips, and to a lesser extent from the intermediate region, of young plants. Disaccharides were predominantly utilized by isolates from plants in the vegetative stage. Overall, the results indicated that growth rates and carbon source utilization reflect the adaptation of bacteria to the rhizosphere environment. The possibility of using these characteristics to screen for rhizosphere competent biocontrol agents that compete for substrates with P. aphanidermatum is discussed.

A nonpathogenic isolate of Fusarium oxysporum, 618-12, added to soil prior to the pathogen, suppressed fusarium wilt (F. o. f.sp. dianthi race 2) in a susceptible cultivar of carnation by 80% compared with the treatment with the pathogen only. The possibility of systemically induced resistance by the nonpathogenic isolate was assessed by inoculating antagonist and pathogen at different locations (stem versus soil, soil versus stem, and in a split-root system). No significant disease suppression was found with any of these spatially separated inoculations. However, inoculation of antagonist and pathogen at the same location within the stem (i.e., mixed stem inoculation) resulted in significant and reproducible disease reductions compared with stem inoculation with the pathogen alone. This reduction was found for different inoculum densities and different cultivars. Several other nonpathogenic Fusarium isolates could also reduce wilt symptoms in the susceptible carnation cultivar after mixed stem inoculation with the pathogen. This disease-suppressive effect after mixed stem inoculations may be caused by locally induced resistance or competition between isolates within the stem. Plants showed vascular browning around the inoculation point following inoculation with nonpathogenic isolates. Disease suppression, as well as vascular browning, were absent when dead conidia of the isolate 618-12 were used. After its addition to soil, the isolate was recovered from 44–78% of carnation stems. Spread of the nonpathogenic isolate within the stem occurred only in the first 4 days after stem inoculation, and it remained confined to limited distances from the inoculation point between 4 and 59 days after inoculation. The pathogenic isolate could be isolated at increasingly greater distances from the inoculation point during this period. These data suggest that the nonpathogenic isolate is not actively spread through the plant by growth of the fungus. Keywords: biological control, competition, induced resistance, wilt.

Two nonpathogenic isolates of Fusarium oxysporum were examined for their ability to counteract F. solani f.sp. pisi, which causes foot and dry root rot in pea. Antagonism was studied in vitro, in a sterilized field soil, and in six natural field soils. Besides native F. solani, other typical pea root rot pathogens occurred in the natural field soils. Both nonpathogenic F. oxysporum isolates reduced disease severity and prevented the plant weight losses that occurred owing to F. solani f.sp. pisi in sterilized soil. Precolonization of sterilized soil with nonpathogenic isolates increased the antagonistic effect. Also, in highly infested field soils the addition of nonpathogenic isolates resulted in lower disease severities and higher yields. Colonization of the soil organic matter by F. oxysporum reached 100% in sterilized soil, independent of the presence of F. solani, and 40 – 90% in naturally infested soils containing native F. solani. The performance of benomyl-resistant mutants of F. oxysporum did not differ from their wild types. Key words: antagonism, soil organic matter, colonization, Pisum sativum.

International audience; The development of new biocontrol products against plant diseases requires screening of high numbers of candidate antagonists. Antagonists for commercial use have to fulfill many different requirements. Besides being active against the specific targeted plant pathogens they must be safe and cost-effective. Important criteria besides pathogenic efficacy are market size, ecological characteristics, production costs, safety, environmental risks, and possibilities for intellectual property protection. A stepwise screening program considering these very different aspects is proposed.

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

Bacteria with the ability to solubilize phosphorus (P) and to improve plant health were selected and tested for growth and survival in P-rich animal bone charcoal (ABC). ABC is suggested to be suitable as a carrier for biocontrol agents, offering them a protected niche as well as delivering phosphate to plants, meanwhile re-using P from waste of the food chain. Ninety-seven bacterial isolates from different soils were tested for their potential to dissolve P from ABC. Of these isolates, 60% showed positive scores; they belonged to the genera Arthrobacter, Bacillus, Burkholderia, Collimonas, Paenibacillus, Pseudomonas, Serratia, and Streptomyces. Twelve isolates from different taxonomic groups were selected for further research on growth ability and survival in ABC, and on their potential to control plant pathogens. The highest concentrations of P were dissolved by Pseudomonas chlororaphis and Bacillus pumilus, followed by Paenibacillus polymyxa, Burkholderia pyrrocinia and three Streptomyces isolates. P. chlororaphis and P. polymyxa showed strongest growth inhibition of plant pathogenic Pythium and Fusarium sp., followed by the Streptomyces spp. isolates. Research highlights Describes a stepwise selection procedure with the aim to find beneficial bacteria that can grow in animal bone charcoal (ABC). This research was part of an EU project, with the aim to recycle and upgrade waste from the food chain. The results show that several interesting beneficial bacteria could proliferate and survive in this ABC carrier. Keywords: Biological control; Phosphorus mobilization; Antagonistic bacteria; Animal bone charcoal; Pythium aphanidermatum; Fusarium oxysporum f.sp. radicis lycopersici

Aims: To develop a strain-specific TaqMan® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain-specific sequence selected using REP-PCR (repetitive extragenic palindromic-polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10-fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan® PCR method is a strain-specific real-time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy

Four Gram-negative bacterial strains, recovered from clay soils cultivated with different crops in the Netherland, were subjected to a polyphasic taxonomic study in order to clarify their taxonomic status. Comparative analysis of the 16S rRNA gene sequences revealed that they belong to the genus Lysobacter and to be highly related to the type strains of L. antibioticus DSM 2044(T), L. gummosus DSM 6980(T), and L. capsici DSM 19286(T), displaying 99.1-99.3%, 99.2-99.6% and 99.4-100% sequence similarities, respectively, to these species. The results of DNA-DNA hybridization studies unambigiously indicated that the four strains belonged to the species L. capsici. Nevertheless, DNA fingerprinting and phenotypic characterization indicated that there was a considerable diversification and niche differentiation among the strains belonging to L. capsici. The newly identified L. capsici strains strongly inhibit Rhizoctonia solani AG2 and originate from Rhizoctonia-suppressive soils where also populations of L. antibioticus and L. gummosus were present. This is the first report of the presence of combined populations of closely related Lysobacter spp. within agricultural soils.

Avoidance of plant diseases has been a major driver for the development of soilless cultivation systems. Nevertheless, diseases still occur in these systems and the need for additional control measures exist. Traditionally, control has relied on the use of chemical fungicides but environmental pressure to reduce chemical usage in the environment, and fewer active ingredients registered for use, has stimulated the development of biological methods of disease control. One approach has been to utilise microbial inoculants as straight replacements for chemical pesticides and some commercial products are now available. Sufficient root colonization and activity are key issues for effective biocontrol. Another approach has been to create growing systems with improved suppressiveness towards plant diseases. The challenge is to combine the available strategies into environmentally and economically sound soilless plant production systems with low risks for pathogen outbreaks. Soilless systems have the potential of creating a balance between a pathogen-free start and a suppressive microflora. Keywords Biological control - Disease-suppressive substrate - Hydroponics - Recirculated nutrient solution - Microbial populations - Rhizosphere - Root pathogens

The quality of torrefied grass fibers (TGF) as a new potting soil ingredient was tested in a greenhouse experiment. TGF was colonized with previously selected microorganisms. Four colonization treatments were compared: (1) no inoculants, (2) the fungus Coniochaeta ligniaria F/TGF15 alone, (3) the fungus followed by inoculation with two selected bacteria, and (4) the fungus with seven selected bacteria. Cultivation-based and DNA-based methods, i.e., PCR-DGGE and BOX-PCR, were applied to assess the bacterial and fungal communities established in the TGF. Although colonization was not performed under sterile conditions, all inoculated strains were recovered from TGF up to 26 days incubation. Stable fungal and bacterial populations of 10(8) and 10(9) CFU/g TGF, respectively, were reached. As a side effect of the torrefaction process that aimed at the chemical stabilization of grass fibers, potentially phytotoxic compounds were generated. These phytotoxic compounds were cold-extracted from the fibers and analyzed by gas chromatography mass spectrometry. Four of 15 target compounds that had previously been found in the extract of TGF were encountered, namely phenol, 2-methoxyphenol, benzopyran-2-one, and tetrahydro-5,6,7,7a-benzofuranone. The concentration of these compounds decreased significantly during incubation. The colonized TGF was mixed with peat (P) in a range of 100%:0%, 50%:50%, 20%:80%, and 0%:100% TGF/P (w/w), respectively, to assess suitability for plant growth. Germination of tomato seeds was assessed three times, i.e., with inoculated TGF that had been incubated for 12, 21, and 26 days. In these tests, 90-100% of the seeds germinated in 50%:50% and 20%:80% TGF/P, whereas on average only 50% of the seeds germinated in pure TGF. Germination was not improved by the microbial inoculants. However, plant fresh weight as well as leaf area of 28-day-old tomato plants were significantly increased in all treatments where C. ligniaria F/TGF15 was inoculated compared to the control treatment without microbial inoculants. Colonization with C. ligniaria also protected the substrate from uncontrolled colonization by other fungi. The excellent colonization of TGF by the selected plant-health promoting bacteria in combination with the fungus C. ligniaria offers the possibility to create disease suppressive substrate, meanwhile replacing 20% to 50% of peat in potting soil by TGF.

This study aimed to assess the colonization of thermally treated (i.e. torrefied) grass fibers (TGFs), a new prospective ingredient of potting soil. Eleven bacterial strains and one fungus, Coniochaeta ligniaria F/TGF15, all isolated from TGF or its extract after inoculation with a soil microbial community, were tested for their ability to colonize TGF. Surprisingly, none of these bacteria were able to directly colonize TGF either as single inoculants or as a consortium. Furthermore, bacterial persistence or growth in TGF was not improved by the addition of nutrients or a surfactant. Only extensive washing of the substrate, presumably removing bacteriostatic or bactericidal compounds, allowed bacterial growth on the fibers. Strikingly, the fungal strain consistently colonized TGF up to high densities (up to 10(10) CFU per g dry TGF). Given the unique capacity of this fungus to degrade toxic compounds including phenols, TGF was colonized with it for different periods of time, after which a consortium of seven selected bacterial isolates was added. Co-presence of the fungus, or 3 and 24 h pre-colonization with it, was insufficient to create a habitable environment for the bacterial consortium. However, fungal pre-colonization of minimally 3 days allowed the bacterial consortium to colonize the TGF at numbers up to 10(9) to 10(10) CFU per g dry substrate. The resultant bacterial community consisted of at least four strains, i.e. Pseudomonas putida 15/TGE5, Serratia plymuthica 23/TGE5, Pseudomonas corrugata 31/TGE5, and Methylobacterium radiotolerans 56/TGF10, as shown by PCR of colonies on plates and PCR-DGGE profiling. Two persisters, S. plymuthica 23/TGE5 and P. corrugata 31/TGES, were highly antagonistic towards several phytopathogenic fungi. Thus, a microbial community with plant-beneficial potential was established on TGF, provided that the fungus C. ligniaria F/TGF15 first creates habitable space in the matrix. (C) 2008 Elsevier B.V. All rights reserved.

Pythium aphanidermatum (Edson) Fitzp., causing root and crown rot in cucumber, was successfully managed by Lysobacter enzymogenes strain 3.1T8. Greenhouse experiments were performed with cucumber plants grown in rockwool blocks up to 5 weeks with a recirculated nutrient solution. Application of L. enzymogenes 3.1T8 in combination with chitosan (the deacetylated derivative of chitin) reduced the number of diseased plants by 50–100% in four independent experiments relative to the Pythium control. Application of chitosan or the bacterial inoculant alone was not effective. Washed bacterial cells plus chitosan inhibited Pythium-induced disease, but the supernatant without bacterial cells combined with chitosan was not effective. The most effective and convenient type of commercially available chitosan was selected. Chitosan disappeared from the hydroponic system within 24 h after application, which we attribute to enzyme expression of L. enzymogenes 3.1T8 induced by the exposure to chitosan. Plate counts of the nutrient solution on a general bacterial medium showed the dominance of the inoculated strain, and an increased bacterial population growing on chitin and chitosan as single carbon source. The population density of L. enzymogenes 3.1T8 on the cucumber roots was investigated with a strain specific real-time TaqMan PCR. Highest chitosan concentrations applied (0.1 and 0.03 g/plant) resulted in the highest numbers of L. enzymogenes 3.1T8 present on roots; i.e. 108–109 cells/g root. Substantially higher numbers of bacterial cells were observed by scanning electron microscopy after application of chitosan; no morphological or other qualitative differences were found. The results indicate that addition of chitosan enhanced the biocontrol efficacy of L. enzymogenes 3.1T8; either chitosan serves as C- and N-source for the antagonist, induces antagonistic gene expression, or both. Keywords: Biological control; Lysobacter enzymogenes; Chitosan; Synergistic effect; Quantitative PCR; Root colonization

The capacity of the microflora in rockwool to suppress Pythium aphanidermatum, the causative agent of root and crown rot in cucumber, was assessed. Disease development of cucumber (Cucumis sativus) grown on rockwool was evaluated in an “ebb-and-flood” system with a recirculating nutrient solution after inoculation with P. aphanidermatum. In five independent experiments from 1995 to 1998, 11 batches of used rockwool were tested. All batches without P. aphanidermatum problems in the preceding cucumber crop had significantly lower numbers of diseased plants in nonautoclaved than in autoclaved used rockwool; the disease incidence was reduced by 52 to 100%. Suppressiveness also was present in rockwool previously used to grow other vegetable crops. Rockwool originating from a cucumber crop that was severely attacked by Pythium resulted in a high disease incidence. Previously unused (new) rockwool had higher or similar percentages of diseased plants than did nonsterilized used rockwool. Disease suppression in used rockwool could also be measured in a smaller test system. In both systems, autoclaved rockwool became suppressive to Pythium after recolonization with the indigenous microflora. Population sizes of total culturable aerobic bacteria as well as of fluorescent pseudomonads did not correlate with disease suppressiveness, as numbers of bacteria and pseudomonads were similar or lower in nonautoclaved (suppressive) than in autoclaved (nonsuppressive) rockwool. Differences in the structure of the bacterial populations could be visualized by using eubacterial polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE). Interestingly, the nonautoclaved and the recolonized used rockwool, which were both suppressive, showed different dominating bacterial groups as compared with the autoclaved rockwool. PCR-DGGE patterns obtained at different sampling times showed that the composition of the bacterial populations changed during plant growth. Fungal populations were present in the treatments that yielded suppressive rockwool, i.e., the nonautoclaved and the recolonized rockwool, but they were absent or present in low numbers in the autoclaved rockwool, which permitted a high disease incidence. Suppressiveness of rockwool to Pythium root and crown rot is a hitherto undescribed phenomenon, and knowledge of the mechanism and microorganisms involved will stimulate the development of microbially balanced soilless growing systems.