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Background Microbes benefit plants by increasing nutrient availability, producing plant growth hormones, and protecting against pathogens. However, it is largely unknown how plants change root microbial communities. Results In this study, we used a multi-cycle selection system and infection by the soilborne fungal pathogen Rhizoctonia solani AG8 (hereafter AG8) to examine how plants impact the rhizosphere bacterial community and recruit beneficial microorganisms to suppress soilborne fungal pathogens and promote plant growth. Successive plantings dramatically enhanced disease suppression on susceptible wheat cultivars to AG8 in the greenhouse. Accordingly, analysis of the rhizosphere soil microbial community using deep sequencing of 16S rRNA genes revealed distinct bacterial community profiles assembled over successive wheat plantings. Moreover, the cluster of bacterial communities formed from the AG8-infected rhizosphere was distinct from those without AG8 infection. Interestingly, the bacterial communities from the rhizosphere with the lowest wheat root disease gradually separated from those with the worst wheat root disease over planting cycles. Successive monocultures and application of AG8 increased the abundance of some bacterial genera which have potential antagonistic activities, such as Chitinophaga, Pseudomonas, Chryseobacterium, and Flavobacterium, and a group of plant growth-promoting (PGP) and nitrogen-fixing microbes, including Pedobacter, Variovorax, and Rhizobium. Furthermore, 47 bacteria isolates belong to 35 species were isolated. Among them, eleven and five exhibited antagonistic activities to AG8 and Rhizoctonia oryzae in vitro, respectively. Notably, Janthinobacterium displayed broad antagonism against the soilborne pathogens Pythium ultimum, AG8, and R. oryzae in vitro, and disease suppressive activity to AG8 in soil. Conclusions Our results demonstrated that successive wheat plantings and pathogen infection can shape the rhizosphere microbial communities and specifically accumulate a group of beneficial microbes. Our findings suggest that soil community selection may offer the potential for addressing agronomic concerns associated with plant diseases and crop productivity.

Actinomycetes are considered to be the biggest producer of bioactive compounds which are expected to have antifungal activity for controlling many fungi such as Rhizoctonia solani. The objective of this study was to obtain potential soybean rhizosphere actinomycetes as a biocontrol agent for R. solani which cause damping-off disease both in vitro and in vivo, including their ability to produce siderophore, chitinase, and HCN. Out of 26 isolates, 18 (56%) showed diverse antifungal activities against R. solani with percentages of inhibition radial growth (PIRG) from 18.9 to 64.8%, as evaluated by a dual culture method. Ten isolates with the strongest antifungal activity were numbered for further characterization. All the tested isolates were not antagonistic towards Bradyrhizobium japonicum. These isolates were able to suppress damping-off disease caused by R. solani in the greenhouse experiment. Isolate ASR53 showed the highest disease suppression, 68% and 91% in sterile and non-sterile soil, respectively. Based on 16S rRNA sequence analysis this isolate belonged to Streptomyces violaceorubidus LMG 20319 (similarity 98.8%) according to GenBank data base available at www.ncbi.nlm.gov.nih. Furthermore, isolate ASR53 had significantly longer roots and shoots, as well as greater fresh and dry weights of seedlings than the control. Crude extract derived from ASR53 isolates contained 10 dominant compounds that were biologically active against fungal pathogens. Thus, this study suggests that the application of potential actinomycetes of the soybean rhizosphere can act as a promising biocontrol agent against damping-off disease caused by R. solani.

Background The development of suppressive soils is a promising strategy to protect plants against soil-borne diseases in a sustainable and viable manner. The use of crop rotation and the incorporation of plant residues into the soil are known to alleviate the stress imposed by soil pathogens through dynamics changes in soil biological and physicochemical properties. However, relatively little is known about the extent to which specific soil amendments of plant residues trigger the development of plant-protective microbiomes. Here, we investigated how the incorporation of pineapple residues in soils highly infested with the banana Fusarium wilt disease alleviates the pathogen pressure via changes in soil microbiomes. Results The addition of above- and below-ground pineapple residues in highly infested soils significantly reduced the number of pathogens in the soil, thus resulting in a lower disease incidence. The development of suppressive soils was mostly related to trackable changes in specific fungal taxa affiliated with Aspergillus fumigatus and Fusarium solani, both of which displayed inhibitory effects against the pathogen. These antagonistic effects were further validated using an in vitro assay in which the pathogen control was related to growth inhibition via directly secreted antimicrobial substances and indirect interspecific competition for nutrients. The disease suppressive potential of these fungal strains was later validated using microbial inoculation in a well-controlled pot experiment. Conclusions These results mechanistically demonstrated how the incorporation of specific plant residues into the soil induces trackable changes in the soil microbiome with direct implications for disease suppression. The incorporation of pineapple residues in the soil alleviated the pathogen pressure by increasing the relative abundance of antagonistic fungal taxa causing a negative effect on pathogen growth and disease incidence. Taken together, this study provides a successful example of how specific agricultural management strategies can be used to manipulate the soil microbiome towards the development of suppressive soils against economically important soil-borne diseases.

The aim of this study was to assess the ability of Bacillus amyloliquefaciens, to augment plant growth and suppress gray mold and leaf spot in pepper plants. Morphological modifications in fungal pathogen hyphae that expanded toward the PGPR colonies were detected via scanning electron microscope. Furthermore, preliminary screening showed that PGPR could produce various hydrolytic enzymes in its media. Treatments with B. amyloliquefaciens suppressed Botrytis gray mold and Alternaria leaf spot diseases on pepper caused by Botrytis pelargonii and Alternaria alternata, respectively. The PGPR strain modulated plant physio-biochemical processes. The inoculation of pepper with PGPR decreased protein, amino acid, antioxidant, hydrogen peroxide, lipid peroxidation, and abscisic acid levels but increased salicylic acid and sugar levels compared to those of uninoculated plants, indicating a mitigation of the adverse effects of biotic stress. Moreover, gene expression studies confirmed physio-biochemical findings. PGPR inoculation led to increased expression of the CaXTH genes and decreased expression of CaAMP1, CaPR1, CaDEF1, CaWRKY2, CaBI-1, CaASRF1, CaSBP11, and CaBiP genes. Considering its beneficial effects, the inoculation of B. amyloliquefaciens can be proposed as an eco-friendly alternative to synthetic chemical fungicides.

Biological control of Rhizoctonia root rot and growth-promoting potential on two tomato cultivars was performed using Pythium oligandrum in combination, or not, with three tomato-associated rhizobacterial strains of Bacillus subtilis, B. thuringiensis and Enterobacter cloacae. The three bacterial strains displayed antifungal activity against Rhizoctonia solani, with pathogen growth inhibition up to 60%. P. oligandrum also destroyed R. solani cells by antibiosis and mycoparasitism processes. In a three-month greenhouse assays, for all trials and the two tomato cultivars used, disease suppression potential, as compared to the controls, was up to 80% using the microorganisms mixtures and 74% by using P. oligandrum alone. Increased height in disease-free plants was obtained with the microorganism mixtures (59%) compared to P. oligandrum (49%). Plants grown in R. solani inoculated peat and challenged with microorganism mixtures were higher than controls, whereas those amended with P. oligandrum showed 46 to 87% height increase. All treatments with the microorganisms were more effective in suppressing the disease than chemical fungicide. Increment of aerial parts and root fresh weights on disease-free plants were until 51% for those treated with microorganism mixtures compared to 46% recorded using P. oligandrum. On inoculated plants, these parameters were enhanced using the mixed treatment. No differences in the rhizosphere-bacterial populations were observed by Single Strand Conformational Polymorphism (SSCP) when, either the rhizobacteria, with or without P. oligandrum, or the pathogen, were inoculated. This suggests that the native bacterial communities were not significantly impacted by the various microbial-inoculations. Only differences were obtained for the two tomato cultivars that the native bacterial microflora colonize.

Disease-suppressive soils protect plants against soilborne fungal pathogens that would otherwise cause root infections. Soil suppressiveness is, in most cases, mediated by the antagonistic activity of the microbial community associated with the plant roots. Considering the enormous taxonomic and functional diversity of the root-associated microbiome, identification of the microbial genera and mechanisms underlying this phenotype is challenging. One approach to unravel the underlying mechanisms is to identify metabolic pathways enriched in the disease-suppressive microbial community, in particular, pathways that harbor natural products with antifungal properties. An important class of these natural products includes peptides produced by nonribosomal peptide synthetases (NRPSs). Here, we applied functional amplicon sequencing of NRPS-associated adenylation domains (A domains) to a collection of eight soils that are suppressive or nonsuppressive (i.e., conducive) to Fusarium culmorum, a fungal root pathogen of wheat. To identify functional elements in the root-associated bacterial community, we developed an open-source pipeline, referred to as dom2BGC, for amplicon annotation and putative gene cluster reconstruction through analyzing A domain co-occurrence across samples. We applied this pipeline to rhizosphere communities from four disease-suppressive and four conducive soils and found significant similarities in NRPS repertoires between suppressive soils. Specifically, several siderophore biosynthetic gene clusters were consistently associated with suppressive soils, hinting at competition for iron as a potential mechanism of suppression. Finally, to validate dom2BGC and to allow more unbiased functional metagenomics, we performed 10× metagenomic sequencing of one suppressive soil, leading to the identification of multiple gene clusters potentially associated with the disease-suppressive phenotype. IMPORTANCE Soil-borne plant-pathogenic fungi continue to be a major threat to agriculture and horticulture. The genus Fusarium in particular is one of the most devastating groups of soilborne fungal pathogens for a wide range of crops. Our approach to develop novel sustainable strategies to control this fungal root pathogen is to explore and exploit an effective, yet poorly understood naturally occurring protection, i.e., disease-suppressive soils. After screening 28 agricultural soils, we recently identified four soils that were suppressive to root disease of wheat caused by Fusarium culmorum. We also confirmed, via sterilization and transplantation, that the microbiomes of these soils play a significant role in the suppressive phenotype. By adopting nonribosomal peptide synthetase (NRPS) functional amplicon screening of suppressive and conducive soils, we here show how computationally driven comparative analysis of combined functional amplicon and metagenomic data can unravel putative mechanisms underlying microbiome-associated plant phenotypes.

The use of beneficial microorganisms for the biological control of plant diseases and pests has emerged as a viable alternative to chemical pesticides in agriculture. Traditionally, microbe-based biocontrol strategies for crop protection relied on the application of single microorganisms. However, the design of microbial consortia for improving the reliability of current biological control practices is now a major trend in biotechnology, and it is already being exploited commercially in the context of sustainable agriculture. In the present study, exploiting the microbial library of the biocontrol company Koppert Biological Systems, we designed microbial consortia composed of carefully selected, well-characterized beneficial bacteria and fungi displaying diverse biocontrol modes of action. We compared their ability to control shoot and root pathogens when applied separately or in combination as microbial consortia, and across different application strategies that imply direct microbial antagonism or induced systemic plant resistance. We hypothesized that consortia will be more versatile than the single strains, displaying an extended functionality, as they will be able to control a wider range of plant diseases through diverse mechanisms and application methods. Our results confirmed our hypothesis, revealing that while different individual microorganisms were the most effective in controlling the root pathogen Fusarium oxysporum or the foliar pathogen Botrytis cinerea in tomato, the consortia showed an extended functionality, effectively controlling both pathogens under any of the application schemes, always reaching the same protection levels as the best performing single strains. Our findings illustrate the potential of microbial consortia, composed of carefully selected and compatible beneficial microorganisms, including bacteria and fungi, for the development of stable and versatile biological control products for plant protection against a wider range of diseases., This research has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 765290. OK and RS are supported by Koppert Biological Systems. MP is supported by the grant RTI2018-094350-B-C31 from the Spanish National R&D Plan of the Ministry of Science, Innovation and Universities (MICIU) and the European Regional Development Fund (ERDF).

Background Plant diseases caused by fungal pathogen result in a substantial economic impact on the global food and fruit industry. Application of organic fertilizers supplemented with biocontrol microorganisms (i.e. bioorganic fertilizers) has been shown to improve resistance against plant pathogens at least in part due to impacts on the structure and function of the resident soil microbiome. However, it remains unclear whether such improvements are driven by the specific action of microbial inoculants, microbial populations naturally resident to the organic fertilizer or the physical-chemical properties of the compost substrate. The aim of this study was to seek the ecological mechanisms involved in the disease suppressive activity of bio-organic fertilizers. Results To disentangle the mechanism of bio-organic fertilizer action, we conducted an experiment tracking Fusarium wilt disease of banana and changes in soil microbial communities over three growth seasons in response to the following four treatments: bio-organic fertilizer (containing Bacillus amyloliquefaciens W19), organic fertilizer, sterilized organic fertilizer and sterilized organic fertilizer supplemented with B. amyloliquefaciens W19. We found that sterilized bioorganic fertilizer to which Bacillus was re-inoculated provided a similar degree of disease suppression as the non-sterilized bioorganic fertilizer across cropping seasons. We further observed that disease suppression in these treatments is linked to impacts on the resident soil microbial communities, specifically by leading to increases in specific Pseudomonas spp.. Observed correlations between Bacillus amendment and indigenous Pseudomonas spp. that might underlie pathogen suppression were further studied in laboratory and pot experiments. These studies revealed that specific bacterial taxa synergistically increase biofilm formation and likely acted as a plant-beneficial consortium against the pathogen. Conclusion Together we demonstrate that the action of bioorganic fertilizer is a product of the biocontrol inoculum within the organic amendment and its impact on the resident soil microbiome. This knowledge should help in the design of more efficient biofertilizers designed to promote soil function.

Four different biocontrol agents (BCAs) and two composts have been used in this study to evaluate their efficiency against the Phytophthora blight of zucchini caused by Phytophthora capsici. The effects of the BCAs and composts on the microbiota of the zucchini rhizosphere were examined at the end of experimental trials conducted in 2016 and 2017 on two farms in natural and artificially infested soils. The next generation amplicon sequencing technology and quantitative Polymerase Chain Reaction - qPCR were used as targeted and untargeted approaches, respectively. The abundance of P. capsici, as measured for all the treatments over both years, decreased with respect to the untreated control, and it may be assumed that a direct pathogen-beneficial microorganism interaction occurred. All the treatments reduced the disease incidence by at least 50% in both sites in naturally or artificially infested soil and in at both sampling periods. The disease index reflected the pathogen abundance. The development of microbiota and mycobiota was not affected by the BCAs, thus indicating that the tested treatments did not interact negatively with the microbial communities, which in turn suggested a slow but good adaptation of the biocontrol agents to the environment. The combination of targeted and untargeted approaches may help to understand the effects of different BCAs on the development of Phytophthora capsici.

Plants host a mesmerizing diversity of microbes inside and around their roots, known as the microbiome. The microbiome is composed mostly of fungi, bacteria, oomycetes, and archaea that can be either pathogenic or beneficial for plant health and fitness. To grow healthy, plants need to surveil soil niches around the roots for the detection of pathogenic microbes, and in parallel maximize the services of beneficial microbes in nutrients uptake and growth promotion. Plants employ a palette of mechanisms to modulate their microbiome including structural modifications, the exudation of secondary metabolites and the coordinated action of different defence responses. Here, we review the current understanding on the composition and activity of the root microbiome and how different plant molecules can shape the structure of the root-associated microbial communities. Examples are given on interactions that occur in the rhizosphere between plants and soilborne fungi. We also present some well-established examples of microbiome harnessing to highlight how plants can maximize their fitness by selecting their microbiome. Understanding how plants manipulate their microbiome can aid in the design of next-generation microbial inoculants for targeted disease suppression and enhanced plant growth.

Bacterial wilt caused by Ralstonia solanacearum (RS) has become one of the most destructive plant diseases in agricultural crops. Obtaining effective strains with disease control activity is an urgent prerequisite for biological control efficiency. This study was performed to explore if the root-associated diazotrophic rhizobacteria in ginger plants have any potential to induce resistance to RS. Three bacterial strains named N10, SJN3 and SJN5 were obtained from the rhizosphere soil of ginger root and were further evaluated regarding their high inhibitory activity in the preliminary screening and in vitro antagonistic activity against RS. Inoculation with N10, SJN3 and SJN5 showed 47.61%, 49.33% and 43.15% relative disease incidence, respectively. N10 and SJN5 showed the most successful biocontrol efficacy and reduced disease incidence by 39.98% and 45.61%, respectively. Moreover, N10 and SJN5 promoted root length by 25.5% and 26.6%, shoot dry weight by 12.5% and 20.1% and root dry weight by 15.4% and 17.9%, respectively. According to the 16S rDNA partial sequence, N10, SJN3 and SJN5 belonged to the genus Pantoea, Burkholderia and Arthrobacter, respectively. Fluorescent in situ hybridization assay revealed that SJN5 cells were distributed on the root surface and formed a condensed biofilm. This result suggests that this strain, which shows nitrogen fixation and antagonism toward RS, not only can increase plant growth, but can also inhibit plant diseases. The findings of the present study expand our current understanding on the application of diazotrophs as a bacterial biocontrol agent, especially for Arthrobacter sp.

Serious cucumber fusarium wilt (CFW) caused by the pathogenic fungus Fusarium oxysporum f. sp. cucumerinum (Foc) frequently occurs during continuous cultivation. The soil bacteria colonized in Foc hyphosphere are supposed to be related to Foc performance and cucumber health. In the present study, culture-independent approach was used to examine the Foc hyphae-associated bacterial communities in different cucumber cropping systems. It was found that the assembly of Foc hyphae-associated bacterial communities was mainly affected by the continuous cucumber practice followed by the pathogen colonization. Proteobacteria, Actinobacteria and Bacteroidetes accounted for 89–93% of the Foc hyphae-associated bacteria, and their abundance changed along with cucumber growth and Foc presence. The abundance of dominant Achromobacter was driven by cucumber cultivation, while the abundance of Rhizobium increased with continuous cucumber cultivation but decreased by Foc spiking. Bioassay tests of cultivable bacterial strains showed that the proportion of Rhizobium and Achromobacter played a key role in the occurrence of CFW, i.e. decreased Rhizobium and increased Achromobacter in the bacterial mixtures led to the decline of Foc suppression. This study indicates the relationship of Foc hyphae-associated bacterial community and cropping systems, and reveals the roles of some specific assembly of dominant bacteria colonizing Foc hyphae in CFW suppression.

Soil–borne plant pathogens are among the most important limiting factors for the productivity of agro–ecosystems. Fungistasis is the natural capability of soils to inhibit the germination and growth of soil–borne fungi in the presence of optimal abiotic conditions. The objective of this study was to assess the effects of different soil managements, in terms of soil amendment types and frequency of application, on fungistasis. For this purpose, a microcosm experiment was performed by conditioning a soil with frequent applications of organic matter with contrasting biochemical quality ( i. e ., glucose, alfalfa straw and wheat straw). Thereafter, the fungistasis response was assessed on four fungi ( Aspergillus niger, Botrytis cinerea, Pyrenochaeta lycopersici and Trichoderma harzianum ). Conditioned soils were characterized by measuring microbial activity (soil respiration) and functional diversity using the BIOLOG EcoPlates TM method. Results showed that irrespective of the fungal species and amendment types, frequent applications of organic matter reduced fungistasis relief and shortened the time required for fungistasis restoration. The frequent addition of easily decomposable organic compounds enhanced soil respiration and its specific catabolic capabilities. This study demonstrated that frequent applications of organic matter affected soil fungistasis likely as a result of higher microbial activity and functional diversity.

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.

Creating a crop-heterogeneous system by intraspecific mixtures of different rice varieties can substantially reduce blast diseases. Such variety mixtures provide an ecological approach for effective disease control, maintaining high yields with the minimum fungicide applications. Whether such an approach is universally applicable for random rice variety combinations and what is the variation pattern of the diseases under intercropping still remains unclear. We conducted two-year large-scale field experiments involving 47 rice varieties/lines and 98 variety-combinations to compare the occurrence of rice blast in monoculture and intercropping plots at multiple sites. In the experiments, the plant height of the selected traditional varieties was about 30 cm taller, and their life cycle was 10 days longer, than that of the improved rice varieties. The monoculture included either traditional or modern rice varieties grown in separate plots. The intercropping included both traditional and modern rice varieties planted together in the same plots. Results from the field experiments under natural disease conditions demonstrated significant reduction for rice blast disease in intercropping plots, compared with that in monoculture plots. For traditional varieties, the average blast incidence reduced from ~26% in monoculture to ~10% in intercropping, and the disease severity reduced from ~17 in monoculture to ~5 in intercropping. For modern varieties, the average blast incidence reduced from ~19% in monoculture to ~10% in intercropping, and the severity from ~10 in monoculture to ~4 in intercropping. Traditional rice varieties (~72%) had a much greater increase in the efficiency of disease control than modern varieties (~60%). In addition, substantially lower values of variance in the blast incidence and severity was detected among the variety combinations in intercropping plots than in monoculture plots. Based on these results, we conclude that the intercropping or mixture of rice varieties greatly reduces the occurrence and variation of rice blast disease in particular variety combinations, which makes the intercropping system more stable and consistent for disease suppression on a large scale of rice cultivation.

The present study evaluated the fumigant effect and potential mechanisms of Chinese prickly ash seed kernel (PSK) against tobacco black shank caused by Phytophthora nicotianae Breda de Haan. The results showed that PSK fumigant increased the soil organic matter, hydrolysable nitrogen, available potassium, and total phosphorus significantly, while the pH was decreased. Application of 2% PSK promoted plant growth, with higher plant height, root length, and dry weight compared to those in the treatment with no PSK. Both in vitro and in vivo tests indicated that PSK fumigant effectively inhibited mycelial growth of P. nicotianae, and significantly reduced the severity of tobacco black shank. Based on the analysis of volatiles in PSK by headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS), 28 components were identified, among which monoterpenes were the major components (accounting for 79.46% of peak area). Among the six top components, linalool, piperitone, sabinene, and limonene exhibited strong mycelial inhibition of P. nicotianae. Linalool was the most effective with an IC50 value of 18.03 &mu, L/L. Results of GC-MS revealed that the main components of PSK extract were cis-Vaccenic acid (40.81%), n-Hexadecanoic acid (15.67%), 9,12-Octadecadienoic acid (Z,Z)- (15.25%), Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)- (10.32%), cis-9-Hexadecenoic acid (6.21%), and Psilocin (11.75%). Among them, Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)- and 9,12-Octadecadienoic acid (Z,Z)- have an obvious inhibitory effect on P. nicotianae. Additionally, changes in soil bacterial and fungal communities were observed. The relative abundance of some microbes, such as Pseudomonas, Azospirillum, Thermomonas, Glutamicibacter, Dyella, and Trichoderma, increased, which have many beneficial microbes in these genera, indicating that microbial community shift could be one of the important outcomes of the PSK fumigant effect. In conclusion, PSK could be a new bio-fumigation material against plant soilborne pathogens, which can function as both a fungicide and fertilizer.

The soilborne fungus Rhizoctonia solani anastomosis group (AG) 8 is a major pathogen of grain crops resulting in substantial production losses. In the absence of resistant cultivars of wheat or barley, a sustainable and enduring method for disease control may lie in the enhancement of biological disease suppression. Evidence of effective biological control of R. solani AG8 through disease suppression has been well documented at our study site in Avon, South Australia. A comparative metatranscriptomic approach was applied to assess the taxonomic and functional characteristics of the rhizosphere microbiome of wheat plants grown in adjacent fields which are suppressive and non-suppressive to the plant pathogen R. solani AG8. Analysis of 12 rhizosphere metatranscriptomes (six per field) was undertaken using two bioinformatic approaches involving unassembled and assembled reads. Differential expression analysis showed the dominant taxa in the rhizosphere based on mRNA annotation were Arthrobacter spp. and Pseudomonas spp. for non-suppressive samples and Stenotrophomonas spp. and Buttiauxella spp. for the suppressive samples. The assembled metatranscriptome analysis identified more differentially expressed genes than the unassembled analysis in the comparison of suppressive and non-suppressive samples. Suppressive samples showed greater expression of a polyketide cyclase, a terpenoid biosynthesis backbone gene (dxs) and many cold shock proteins (csp). Non-suppressive samples were characterised by greater expression of antibiotic genes such as non-heme chloroperoxidase (cpo) which is involved in pyrrolnitrin synthesis, and phenazine biosynthesis family protein F (phzF) and its transcriptional activator protein (phzR). A large number of genes involved in detoxifying reactive oxygen species (ROS) and superoxide radicals (sod, cat, ahp, bcp, gpx1, trx) were also expressed in the non-suppressive rhizosphere samples most likely in response to the infection of wheat roots by R. solani AG8. Together these results provide new insight into microbial gene expression in the rhizosphere of wheat in soils suppressive and non-suppressive to R. solani AG8. The approach taken and the genes involved in these functions provide direction for future studies to determine more precisely the molecular interplay of plant-microbe-pathogen interactions with the ultimate goal of the development of management options that promote beneficial rhizosphere microflora to reduce R. solani AG8 infection of crops.

Non-cultivated plants carry microbial endophytes that may be used to enhance development and disease resistance of crop species where growth-promoting and protective microbes may have been lost. During seedling establishment, seedlings may be infected by several fungal pathogens that are seed or soil borne. Several species of Fusarium, Pythium and other water moulds cause seed rots during germination. Fusarium blights of seedlings are also very common and significantly affect seedling development. In the present study we screened nine endophytic bacteria isolated from the seeds of invasive Phragmites australis by inoculating onto rice, Bermuda grass (Cynodon dactylon), or annual bluegrass (Poa annua) seeds to evaluate plant growth promotion and protection from disease caused by Fusarium oxysporum. We found that three bacteria belonging to genus Pseudomonas spp. (SLB4-P. fluorescens, SLB6-Pseudomonas sp. and SY1-Pseudomonas sp.) promoted seedling development, including enhancement of root and shoot growth, and stimulation of root hair formation. These bacteria were also found to increase phosphate solubilization in in vitro experiments. Pseudomonas sp. (SY1) significantly protected grass seedlings from Fusarium infection. In co-culture experiments, strain SY1 strongly inhibited fungal pathogens with 85.71% growth inhibition of F. oxysporum, 86.33% growth inhibition of Curvularia sp. and 82.14% growth inhibition of Alternaria sp. Seedlings previously treated with bacteria were found much less infected by F. oxysporum in comparison to non-treated controls. On microscopic observation we found that bacteria appeared to degrade fungal mycelia actively. Metabolite products of strain SY1 in agar were also found to inhibit fungal growth on nutrient media. Pseudomonas sp. (SY1) was found to produce antifungal volatiles. Polymerase chain reaction (PCR) amplification using specific primers for pyrrolnitirin synthesis and HCN (hydrogen cyanide) production suggested presence of genes for both compounds in the genome of SY1. HCN was detected in cultures of SY1. We conclude that microbes from non-cultivated plants may provide disease protection and promote growth of crop plants.

Intensive farming in agriculture raises questions in relation to environmental sustainability and the widespread use of agrochemicals. In the present work, we compare the impact of organic and intensive farming, in connection to the soil suppressiveness against the soilborne pathogen Rhizoctonia solani. Three farms were considered in the study: two practicing organic cultivation (for 10 and 20 years, respectively), and one applying conventional cultivation. Soil suppressiveness was assessed in a greenhouse bioassay with lettuce (Lactuca sativa). Soil microbiome was characterized by combining BIOLOG EcoPlates™ with high‐throughput sequencing of bacterial and eukaryotic rRNA gene markers. Suppressiveness towards R. solani was higher in organic than in conventional farming soil, but this property was lost after soil sterilization. Functional biodiversity was significantly higher in the two organic soils, and this parameter was predictive of the suppressiveness towards R. solani. According to our analyses, the overall microbial taxonomic diversity was unlinked to suppressiveness. A correlation analysis, carried out at the genus level for the most abundant bacterial and eukaryotic microbial taxa, showed that 58.7% of the genera had a statistically significant correlation with suppressiveness. In particular, the genera Flavisolibacter, Massilia, Pseudomonas, Ramlibacter, Rhizophus and the oligochaete worms belonging to the Enchytraeidae family positively correlated with the disease suppression.

Numerous recent studies have demonstrated that biochar may significantly reduce the incidence of plant diseases caused by airborne and soilborne pathogens, although contrasting results have also been reported. In this work, we investigated how biochar affects crop plant and soilborne microbe growth. Aims of this study were: i) to analyze the chemical changes occurring in four organic feedstocks (e.g. wood chips, organic urban waste, Zea mays residues, and Medicago sativa hay) when pyrolyzed at 300 °C and 550 °C by using 13C NMR spectroscopy and SEM (Scanning Electron Microscopy); ii) to assess how biochar affects growth of five bacteria, nine fungi, and three crop plants; and iii) clarify the relationships between biochar chemistry and its effect on target species. As pyrolyzation temperature increased, organic matter chemistry of all products changed significantly, with a progressive loss of O-alkyl C, di-O-alkyl C, and methoxyl and N-alkyl C, coupled with an enrichment in aromatic C types. Untreated urban waste and Medicago hay severely inhibited Lepidium, Lactuca and Solanum root growth, whereas no inhibitory effects were found for the other feedstocks. However, these phytotoxic effects largely decreased after pyrolyzation. In contrast to the crop plants, fungi and bacteria thrive on most of the unprocessed organic materials but showed reduced growth and development or complete growth inhibition on biochars obtained at 300 °C and 550 °C. Soilborne microbes demonstrated remarkably similar correlation patterns between their growth to the organic feedstock and biochar chemical components. This work demonstrates that defining organic matter quality by 13C NMR extends our understanding of the impact of biochar on crop plants and key components of the soil food-web.

Suppressive composts represent a sustainable approach to combat soilborne plant pathogens and an alternative to the ineffective chemical fungicides used against those. Nevertheless, suppressiveness to plant pathogens and reliability of composts are often inconsistent with unpredictable effects. While suppressiveness is usually attributed to the compost's microorganisms, the mechanisms governing microbial recruitment by the roots and the composition of selected microbial communities are not fully elucidated. Herein, the purpose of the study was to evaluate the impact of a compost on tomato plant growth and its suppressiveness against Fusarium oxysporum f. sp. lycopersici (Foxl) and Verticillium dahliae (Vd). First, growth parameters of tomato plants grown in sterile peat-based substrates including 20 and 30% sterile compost (80P/20C-ST and 70P/30C-ST) or non-sterile compost (80P/20C and 70P/30C) were evaluated in a growth room experiment. Plant height, total leaf surface, and fresh and dry weight of plants grown in the non-sterile compost mixes were increased compared to the plants grown in the sterile compost substrates, indicating the plant growth promoting activity of the compost's microorganisms. Subsequently, compost's suppressiveness against Foxl and Vd was evaluated with pathogenicity experiments on tomato plants grown in 70P/30C-ST and 70P/30C substrates. Disease intensity was significantly less in plants grown in the non-sterile compost than in those grown in the sterile compost substrate; AUDPC was 2.3- and 1.4-fold less for Foxl and Vd, respectively. Moreover, fungal quantification in planta demonstrated reduced colonization in plants grown in the non-sterile mixture. To further investigate these findings, we characterized the culturable microbiome attracted by the roots compared to the unplanted compost. Bacteria and fungi isolated from unplanted compost and the rhizosphere of plants were sequence-identified. Community-level analysis revealed differential microbial communities between the compost and the rhizosphere, suggesting a clear effect of the plant in the microbiome assembly. Proteobacteria and Actinobacteria were highly enriched in the rhizosphere whereas Firmicutes were strongly represented in both compartments with Bacillus being the most abundant species. Our results shed light on the composition of a microbial consortium that could protect plants against the wilt pathogens of tomato and improve plant overall health.

A novel strategy for controlling the spread of arboviral diseases such as dengue, Zika and chikungunya is to transform mosquito populations with virus-suppressingWolbaehia. In general,Wolbachiatransinfected into mosquitoes induce fitness costs through lower viability or fecundity. These maternally inherited bacteria also produce a frequency-dependent advantage for infected females by inducing cytoplasmic incompatibility (CI), which kills the embryos produced by uninfected females mated to infected males. These competing effects, a frequency-dependent advantage and frequency-independent costs, produce bistableWolbachiafrequency dynamics. Above a threshold frequency, denotedp̂, CI drives fitness-decreasingWolbachiatransinfections through local populations; but belowp̂, infection frequencies tend to decline to zero. Ifp̂is not too high, CI also drives spatial spread once infections become established over sufficiently large areas. We illustrate how simple models provide testable predictions concerning the spatial and temporal dynamics ofWolbachiaintroductions, focusing on rate of spatial spread, the shape of spreading waves, and the conditions for initiating spread from local introductions. First, we consider the robustness of diffusion-based predictions to incorporating two important features ofwMel-Aedes aegyptibiology that may be inconsistent with the diffusion approximations, namely fast local dynamics induced by complete CI (i.e., all embryos produced from incompatible crosses die) and long-tailed, non-Gaussian dispersal. With complete CI, our numerical analyses show that long-tailed dispersal changes wave-width predictions only slightly; but it can significantly reduce wave speed relative to the diffusion prediction; it also allows smaller local introductions to initiate spatial spread. Second, we use approximations forp̂and dispersal distances to predict the outcome of 2013 releases ofwMel-infectedAedes aegyptiin Cairns, Australia, Third, we describe new data fromAedes aegyptipopulations near Cairns, Australia that demonstrate long-distance dispersal and provide an approximate lower bound onp̂forwMel in northeastern Australia. Finally, we apply our analyses to produce operational guidelines for efficient transformation of vector populations over large areas. We demonstrate that even very slow spatial spread, on the order of 10-20 m/month (as predicted), can produce area-wide population transformation within a few years following initial releases covering about 20-30% of the target area.

International audience; Rhizoctonia solani AG-8 is a major root pathogen in wheat (Triticum aestivum L.) systems worldwide and while natural disease suppression can develop under continuous cropping, this is not always the case. The main aim of our work was to elucidate the rhizosphere microbial community underlying a Rhizoctonia suppressive soil (Avon, South Australia) and to investigate how this community may develop in agricultural soils conducive to disease and of different soil type (Galong and Harden, New South Wales). The Avon suppressive soil community included Asaia spp. and Paenibacillus borealis, which were absent from a paired non-suppressive site. At Galong, soil taken from inside and outside disease patches showed no evidence of suppression, and disease suppression could not be transferred from the suppressive soil to the conducive soil from a different soil type and climatic area. 16S rRNA microarray analysis revealed Pseudomonas spp. were significantly more abundant inside than outside three disease patches at Galong. However, a survey of 32 patches across a range of stubble and tillage treatments at a nearby site showed no correlation between Pseudomonas and disease incidence. R. solani levels were significantly lower when stubble was retained rather than burnt or when nutrients (N, P and S) were incorporated with stubble during the non-crop period. Our results suggest soil type is an important factor for suppressive capability and that where specific disease suppression is absent, agronomic practice to increase soil carbon can encourage a non-specific microbial response that limits disease severity.

A field experiment was conducted to compare effects of four types of organic amendments on soil chemical, microbiological and disease suppression characteristics in an organic farm. The amendments were plant-derived fresh compost (C), steer-derived slurry (S), slurry plus dung (SD) and slurry, compost and dung (SCD). We hypothesized that amendments with more easily available carbon sources (S and C) might enhance Fusarium wilt of flax, while more complex amendments with lower carbon availability might suppress the disease. Fusarium wilt development was tested in bioassays with flax growing in Fusarium oxysporum f. sp. lini Snyder & Hansen inoculated soil samples from previously amended plots in a growth chamber, and area under the disease progress curves (AUDPCs) were determined per pot. Soil chemical and microbial analyses were conducted directly on soil samples taken from the field. Total DNA was extracted from soil and amplified with specific primers for eubacteria, Pseudomonas species, ammonia-oxidizing bacteria (AOB) and fungi. The respective microbial compositions were assessed by denaturing gradient gel electrophoresis, and species richness and diversity were calculated from the numbers and intensities of the amplicons on the gels. Fusarium wilt was significantly suppressed in soil from SCD treated plots and enhanced in that from C-treated plots. AUDPC values were negatively correlated with pH only. In stepwise and canonical discriminant analyses soil samples with low and high AUDPC values were distinguished based on pH, total carbon content, and diversity of AOB. It is suggested that AOB could be useful indicators for suppression of soil-borne pathogens.

The need for studying tailored composts, as new feedstocks become available with time, increases the in-depth studies of suppressive composts derived from green sources and agro-wastes recycling. The composition, diversity and variability of microbiomes within a collection of 10 composts were investigated by amplicon sequencing and bioinformatics analyses. The observed differences in microbiome structure were related to the different compost origin. The multi-suppressive properties of composts from agricultural residues, agro-industrial co/by-products, and plant green-waste showed the most complex microbiome structure, which included either biocontrol agents associated with the control of Rhizoctonia damping-off in bean and Verticillium wilt in eggplant or microbial consortia for controlling Pythium damping-off in cucumber and zucchini and Phytophthora root rot in tomato and azalea. In contrast, the pathogen-specific property of composts from municipal solid waste and co-composted cow manure household waste showed a microbiome that overall included biocontrol agents against Fusarium wilt in tomato, melon and basil. The highest correlations between physicochemical properties, disease suppression, and the microbiome have allowed to make a helpful matrix to know how relationships among these variables of composts could be established and quantified for predicting their suppressive properties basing on the physicochemical properties and the microbiome.

Plant growth promoting rhizobacteria (PGPR) are associated with plant roots and augment plant productivity and immunity by reducing fertilizer application rates and nutrient runoff. Studies were conducted to evaluate bell pepper transplants amended with formulation of consortium of two indigenous PGPR isolates (Bacillus subtilis and Bacillus pumilus) in terms of increase in yield and disease resistance under field conditions. Transplants were planted into plots treated by NPK (nitrogen, phosphorus and potassium), fungicides, soil solarization, MeBr fumigation, PGPR and untreated soil. Treatments were assessed for incidence of soil-borne phytopathogens viz. Phytophthora capsici and Colletotrichum sp. Highly significant increases in bell pepper transplant growth occurred in response to formulations of PGPR isolates. Transplant vigor and survival in the field were also improved by PGPR treatments. Consortium of Bacillus subtilis and Bacillus pumilus reduced disease incidence of damping off by 1.81% and anthracnose by 1.75%. Numbers of colony forming units of Phytophthora capsici and Colletotrichum sp. were significantly higher in all plots than those treated with PGPR consortium. Incidence of seed rot and seedling blight on bell pepper was significantly lower in PGPR-treated plots and highest in untreated plots. Total fruit yield of bell pepper increased by 379.36% with PGPR consortium (Bacillus subtilis and Bacillus pumilus).

Plant growth promoting rhizobacteria (PGPR) are the rhizosphere bacteria that may be utilized to augment plant growth and suppress plant diseases. The objectives of this study were to identify and characterize PGPR indigenous to cucumber rhizosphere in Bangladesh, and to evaluate their ability to suppress Phytophthora crown rot in cucumber. A total of sixty six isolates were isolated, out of which ten (PPB1, PPB2, PPB3, PPB4, PPB5, PPB8, PPB9, PPB10, PPB11 and PPB12) were selected based on their in vitro plant growth promoting attributes and antagonism of phytopathogens. Phylogenetic analysis of 16S rRNA sequences identified these isolates as new strains of Pseudomonas stutzeri, Bacillus subtilis, Stenotrophomonas maltophilia and B. amyloliquefaciens. The selected isolates produced high levels (26.78 to 51.28 μg mL−1) of indole-3-acetic acid, while significant acetylene reduction activities (1.79 to 4.9 µmole C2H4 mg-1 protein h-1) were observed in eight isolates. Cucumber plants grown from seeds that were treated with these PGPR strains displayed significantly higher levels of germination, seedling vigor, growth, and N content in root and shoot tissue compared to non-treated control plants. All selected isolates were able to successfully colonize the cucumber roots. Moreover, treating cucumber seeds with these isolates significantly suppressed Phytophthora crown rot caused by Phytophthora capsici, and characteristic morphological alterations in Ph. capsici hyphae that grew towards PGPR colonies were observed. Since these PGPR inoculants exhibited multiple traits beneficial to the host plants, they may be applied in the development of new, safe, and effective seed treatments as an alternative to chemical fungicides.

Organic and conventional greenhouse compartments were set up to compare tomato production, disease development and microbial communities in two systems side by side. After one year, the organic greenhouse was split into two sections: one where straw was added to the soil (to reduce soil nitrogen) and the other without straw. This paper reports on naturally emerging corky root (CR) disease and root knot nematode (RKN) galling in the second and third years of the experiment. CR increased over the years in all systems, but earlier in the conventional system, so that there were significant differences between the two organic versus the conventional systems in the second year but not anymore in the third year. RKN galling became apparent in the third year, particularly in the conventional system and rarely in the organic systems. CR severity was significantly negatively correlated with dissolved organic carbon content, total carbon, nitrogen concentrations and oxygen uptake rate in soil, and with tomato yield. RKN galling was too rare to be related to any variables. CR severity classes were significantly separated based on selected operational taxonomic units (OTUs) or phylotypes of 16S rDNA for bacteria and actinomycetes and of 18S rDNA ITS for fungi detected by denaturing gradient gel electrophoresis (DGGE). Microbial communities in the rhizoplane were largely subsets of those in the rhizosphere and bulk soil, but the distributions of OTUs over different habitats were similar for the conventional and recently converted organic systems. These results may stimulate research into selection of microbial communities from one habitat into neighboring ones to support the hypothesis of microbial cycling in ecosystems.

Compost teas are fermented extracts of composted materials that are used to control plant diseases. A compost extractor in liquid phase, with forced air blowing system and assembled using farmer facilities, was used to produce “on farm” aerated compost teas (CTs) from five types of compost, in a 14-day fermentation cycle. Solid feed stocks, including one biowaste compost and four composted tomato residues, were extracted in water (waCTs) and whey (whCTs), separately. The ten teas showed high biological control ability, both in vitro and in vivo, against three tomato pathogens: Alternaria alternata, Botrytis cinerea and Pyrenochaeta lycopersici .I nin vitro trials, the suppressive ability of raw CTs was lost with sterilization, suggesting an antibiotic-like antagonism effect due to active microorganisms in suspension. Direct application of teas on tomato plants significantly reduced disease symptoms caused by tested pathogens. In vivo suppressiveness was more significant using the liquid phase fermentation (whey or water) than the compost. Whey could be considered a viable extractant for suppressive compost-tea production, although further dilution in dechlorinated water at a ratio of at least 1:5 proved a necessary method to avoid occurrence of root or foliar phytotoxicity, probably due to high salt concentrations and sub-acid pH of the relative teas.

Organic amendments influence chemical and microbial compositions in soils and also susceptibility to plant diseases. The purpose of this study was to establish different parameters that interfere with pathogen growth in soil. Four different organic-amendment regimes, i.e. slurry, compost, slurry-dung and compost-slurry-dung, were applied to two fields proximate to each other on the same farm and covered with the same grass-clover ley in the last two years preceding sampling. Before that period, there were differences in cropping and management practices between both fields. Chemical analyses of the soils revealed no differences between organic amendments, whereas significant differences were present between fields in all C, N and pH values. Growth of R. solani AG2-2IIIB in soil, measured by damping-off in sugar beet plants, was influenced by the interaction of organic amendment with field type. Diversity and evenness values of the microbial communities, studied by PCR-DGGEs specific for bacteria, fungi, Pseudomonas and ammonia-oxidizing beta-proteobacteria, revealed different patterns, i.e. no differences between organic amendments, but clear differences between fields. Multivariate analyses done on individual species of the four groups, as represented by band location and size in PCR-DGGE fingerprints, and by inclusion of chemical and R. solani AG2-2IIIB growth parameters as `environmental' variables, revealed strong and occasionally significant effects of organic matter content, water-dissolvable organic carbon and pH on microbial communities. It was therefore concluded that different organic amendments had different effects on pathogen growth in soils of both fields and that organic matter content and pH influenced soil microbial compositions most.

Plants are liable to be attacked by soilborne fungal pathogens which are responsible to reduce plant growth and losses in yield. In Indonesia, indigenous soybeans’ rhizobacteria such as antifungal producing Pseudomonas sp. have not many been reported yet. Therefore, the potential of the Pseudomonas sp. as biocontrol agent should be deeply explored. The aim of this study was to screen the indigenous soybeans’ rhizobacteria Pseudomonas sp. that possessing biocontrol characters against soilborne mainly i.e. Sclerotium rolfsii, Fusarium oxysporum, and Rhizoctonia solani, in vitro and in planta. Eleven isolates identified Pseudomonas sp. CRB numbered by CRB-3, CRB-16, CRB-17, CRB-31, CRB-44, CRB-75, CRB-80, CRB-86, CRB-102, CRB-109, and CRB-112 were affirmed to be candidates of biocontrol agents toward the soilborne fungal pathogens. Pseudomonas sp. CRB inhibited growth of the pathogenic fungi approximately 11.1-60.0% in vitro. Among of them, 7 isolates were also produced siderophore, 2 isolates produced chitinase, and 4 isolates produced hydrogen cyanide. Seed coating with the Pseudomonas sp. CRB accomplished disease suppression in planta about 14.3-100% in sterile soil condition and 5.2-52.6% in non sterile soil condition. Consistency in high performance more than 30% of disease suppression in non sterile soil condition suggested that 5 isolates i.e. CRB-16, CRB-44, CRB-86, CRB-102, and CRB-109 isolates have great promising to be developed as biocontrol agents of soilborne pathogenic fungi.

Rhizoctonia solani is a complex species that is composed of different anastomosis groups (AG). Although these different AGs show differences in their host ranges, generally R. solani is a phytopathogenic species with a wide spectrum of hosts. It has the ability to grow as a saprotroph, which further complicates its behaviour as a parasite. The losses caused by R. solani are very important and need a sustainable management strategy. The patchy appearance of the disease caused by this pathogen is well-known. The patches show within and between season dynamics. The factors which affect the spread of the disease can be grouped into three main categories: host plant, pathogen and environment. However, each of the categories in its detail may depend on or react with the other categories. There are a number of factors that may be involved in dynamics of patches. These potential mechanisms are discussed. It is essential to know about the mechanisms involved to develop an effective control strategy. Although more work is needed to investigate different mechanisms of parasitism deployed by different AGs in different hosts, it seems that many mechanisms external to the host are operating at the same time which necessitates an integrative research approach to study and control the diseases caused by R. solani.

The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil. (C) 2008 Elsevier B.V. All rights reserved.

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

A less resource intensive procedure was used to select an endophytic non-pathogenic isolate of Fusarium oxysporum (NP Fo ) and an antibiotic producing rhizosphere/rhizoplane (RS-RP) competent fluorescent pseudomonad to suppress Fusarium yellow ( F. oxysporum (Schlecht) f. sp. phaseoli Kendrick and Snyder) of bean ( Phaseolus vulgaris L.). Using the combined criteria of in vitro and in planta competitiveness resulted in selection of three isolates for determination of their disease suppressing attributes. Several isolates of fluorescent pseudomonads obtained from RS-RP and internal root tissues of bean were tested for antibiosis in vitro. The isolates producing large inhibition zones were selected to determine RS-RP competence through seed treatment. The three most competent isolates were tested for their capacity to reduce root colonization by the pathogen, which resulted in the selection of only one isolate, identified as Pseudomonas putida (Trevisan) Migula biotype A. The NP Fo isolates Fo10 and Fo6 endophytically occupied about 80% of the root length compared to only 60% by the isolate Fo16. Mixing chlamydospores of Fo10 in non-sterile field soil, reduced RS-RP and internal root colonization by the pathogen by 95 and 89%, respectively. In the greenhouse trial, mixing chlamydospores of either of the three NP Fo isolates, the area under the chlorophyll retention curve (AUCRC) increased significantly with Fo10 compared to Fo6 or F16, over the positive control. The grain yield/plants from Fo10 treated soil was 177 or 291% higher than from Fo6-soil or positive control, respectively, but remained 50% lower than in pathogen-free negative control soil. In the field plots with pathogen ID of 850 ± 150/g soil, mixing chlamydospores of Fo10 (4250 ± 113/g), increased AUCRC and grain yield by about 100% over the control. However, if the bean seeds were treated with P. putida , the grain yield increased by 211% without further increase in AUCRC. The suppressive effect of Fo10 on disease development appeared to be related to its high saprophytic competitiveness with the pathogen in the soil matrix, which resulted in lower ID in the root zone, and parasitic competitiveness by reducing the availability of infection sites and internal root colonization by the pathogen. Seed treatment with P. putida provided additional protection in the root zone.

Two species of Pseudomonas chromosomally tagged with gfp, which had shown antagonistic activity against the tomato pathogen Ralstonia solanacearum in a previous study, were assessed for their impact in the rhizosphere of maize. Plant growth characteristics, numbers of indigenous heterotrophic bacteria, changes in the bacterial community structure according to the r/K strategy concept, and shifts in MIDI-FAME profiles of culturable bacterial fractions as well as total rhizosphere microbial communities were determined in relation to seed and soil treatment with the exogenous pseudomonads. The maize rhizosphere proved to be a suitable habitat for the introduced P. chlororaphis IDV1 and P. putida RA2, which showed good survival after introduction. However, both inoculants showed a small growth-reducing effect towards maize, which might have been caused by the high densities of inoculants used (i.e. competition for nutrients and action of metabolites produced) and/or changes in microbial community structure (both culturable bacterial fraction and the total microflora). Probably, an altered balance among the indigenous maize rhizosphere populations occurred. Thus, the culturable bacteria, as well as the total microflora in the rhizosphere, changed in response to the introduced pseudomonads, and their development was dependent on the growth stage of the plant. The FAME analyses showed that these microbial communities comprised different populations, and were separated according to, first, the method used (direct versus cultivation-based), second, sampling time, and, finally, inoculation level. (C) 2004 Elsevier Ltd. All rights reserved.

Previously, we showed that bacterial populations oscillated in a regular manner in response to a nutrient impulse in soil. For this paper we investigated if the wave-like fluctuations in bacterial populations could be explained by their interactions with populations of bacterial-feeding nematodes (BFN). In two microcosm experiments, soil bacterial populations-colony forming units (CFUs) and microscopic counts of stained bacteria and nematode populations in 22 families were monitored daily for 25 or 30 days after incorporation of clover + grass (CG) plant material into soil. In another microcosm experiment, dynamics of bacteria and nematode populations were monitored in response to gamma-irradiated plant material added to gamma-irradiated soil mixed with filtered bacterial suspensions and in non-irradiated soil. In the first experiment, soil bacterial populations fluctuated significantly after incorporation of the plant material with two peaks within the first week and three or four smaller peaks thereafter. Populations of total nematodes and BFN started to increase in the second week after CG incorporation, but the proportion of BFN increased within 1 week. Inactive juvenile BFN (dauerlarvae) seemed to be activated after 2 days (as the percentage of Rhabditidae increased and dauerlarvae decreased), followed by stepwise increases in dauerlarvae every 4 days, indicating that there was a new generation every 4 days. There were significant wave-like fluctuations in daily population changes of BFN, but not for those of total nematode communities, over the duration of these experiments. These fluctuations had similar periods (5 days) as those of bacterial populations, but were shifted about 3 days relative to the bacterial fluctuations. Gamma-irradiation of soil significantly increased the periods and amplitudes of bacterial oscillations. Nematode populations were eliminated in gamma-irradiated soils, but small numbers of protozoa were accidentally introduced in the irradiated soil, and may have been partially responsible for the delayed regulation of bacterial growth. We conclude that fluctuations in bacterial populations were not directly related to similar fluctuations in populations of BFN, as expected from classical Lotka-Volterra equations for predator-prey relationships, but were related to changes in growth rates of BFN. An alternation in active and inactive stages in a synchronized predator community after a disturbance could allow periods of bacterial growth alternated with periods of death. Fluctuations in bacterial populations were dampened after a much longer period when the soil fauna was largely eliminated. (C) 2003 Elsevier B.V. All rights reserved.

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

Seed potato production is threatened by potato cyst nematodes (PCN). Seed potatoes can only be grown on PCN-free declared potato fields. A seed potato grower chose inundation, flooding his soil, for controlling PCN because resistant cultivars are economically less advantageous and catch crops or granular fumigants gave insufficient results. Inundation is widely used in bulb cultures in The Netherlands to control parasitic nematodes and selectively also pathogenic fungi. Research was performed on the efficiency of inundation against PCN species Globodera pallida and the causal fungal agent of potato wilt Verticillium dahlia (Vd) in a 30 ha field. The effect on soil fertility and texture was also studied. A technical manual for large-scale inundation was written for growers and economic evaluation was performed. Results showed that after 16 weeks of inundation 99.9% of the contents of artificially applied G. pallida cysts was eliminated. Vitality of V. dahlia was reduced with 84%. Incorporation of B. juncea did not increase efficiency. Anaerobic soil disinfestation (ASD) with incorporated B. juncea in the dike surrounding the field, showed a lower efficiency against G. pallida due to incorrect application. Naturally occurring beet cysts lost their vitality after inundation. Incorporation of B. juncea increased soil suppressiveness against Rhizoctonia solani whereas inundation leveled out this effect. By slow release of water after inundation soil texture remained unchanged. Changes in fertilizer content were affected both by the growth and incorporation of B. juncea and by inundation. An increase of sodium and of the electrical conductivity (EC) was noticed. Inundation proved to be highly effective in controlling PCN and Vd and economically feasible for practical application. Long-term effects have to be monitored and effects on other soil quality aspects, both biological, chemical and physical should be taken into account in future research. For some regions in The Netherlands inundation is a new tool for seed potato growers in their Nematode Control Strategy.

To reduce Fusarium Basal Rot caused by Fusarium oxysporum f.sp. cepae (Foc) through crop rotation, plant species should be selected based on Foc multiplication in their roots. Foc multiplication rates in 13 plant species were tested in a greenhouse. All plant species enabled Foc multiplication. The lowest Foc levels (cfu g−1 dry root) were found for wheat, sunflower, cowpea and millet, the highest for black bean. The highest Foc levels per plant were calculated for sudan grass. These data were used to calibrate the model Pf = Pi/(α + βPi) relating final (Pf) and initial (Pi) Foc levels in the soil. The rate of population increase at low Pi (1/α) was highest for onion and black oat and smallest for sunflower. The pathogen carrying capacity (1/β) was highest for black oat and black bean, and lowest for wheat, cowpea and foxtail millet. Foc soil population dynamics was simulated for crop sequences by concatenating Pi-Pf values, considering instantaneous or gradual pathogen release after harvest. Different soil Foc populations were attained after reaching steady states. Foc populations in the sequence onion –foxtail millet - wheat – cowpea were 67 % lower than in the sequence onion – sudan grass - black oat - black beans. In this work, by combining detailed greenhouse experiments with modelling, we were able to screen crops for their ability to increase Foc population and to explore potential crop sequences that may limit pathogen build-up.

Bacterial populations (CFU) have been shown to oscillate in wavelike patterns after nutrient impulses in previous studies. The amplitudes and periods of oscillations could possibly be used as indicators of soil health analogous to the stability and resilience of biological populations widely accepted as indicators for ecosystem health. Limited plant and animal disease outbreaks can also be viewed as a manifestation of a healthy soil ecosystem. Two pot experiments were carried out to verify whether damping-off of beet seedlings by Pythium ultimum, measured as area under the disease progress curve (AUDPC), fluctuated over time after incorporation of organic materials into organic versus conventional soils, and to investigate whether daily dynamics of AUDPCs were linked to the dynamics of microbial populations and chemical parameters. AUDPCs oscillated significantly over time when Pythium bioassays were initiated daily after addition of ground grass and clover shoots (GC) into unplanted soils. Similar oscillations with significant harmonics of AUDPC were also observed in composted manure (CM)-amended soils but with smaller amplitudes than in GC-amended soils. The AUDPC harmonics in amended soils had periods similar to those of CFU of copiotrophic bacteria. Cross-correlation analysis demonstrated that periodic fluctuations of P. ultimum infections (AUDPCs) did not coincide with those of copiotrophic CFU but were shifted in phase. It appears that competition or antagonism from some fast-growing bacteria influenced pathogen infections, because these bacterial populations were growing and dying. Soil chemical variables, including pH, dissolved organic carbon, and NO3−-N, and NH4+-N contents, changed significantly in the initial 7 days after a nutrient impulse into soils. These changes were cross-correlated with copiotrophic CFU with time lags of ≈1 to 2 days but were seldom associated with daily changes in AUDPCs. Organically managed soils always had lower AUDPC ratios of amended to nonamended treatments, indicating that organic materials showed stronger suppressive abilities to P. ultimum in organic than in conventional soils. The oscillations in AUDPCs and copiotrophic CFU in amended organic soil also had smaller amplitudes than in amended conventional soil. These results suggested that organically managed soils had a greater resistance and resilience to the disturbance of the amendments and, therefore, could be considered healthier than conventionally managed soils.

Arable fields of 10 organic farms from different locations in The Netherlands were sampled in three subsequent years. The soil samples were analysed for disease suppressiveness against Rhizoctonia solani AG2.2IIIB in sugar beet, Streptomyces scabies in radish and Verticillium longisporum in oilseed rape. In addition, a variety of microbial, chemical and physical soil characteristics were assessed. All data were correlated by multiple regression and multivariate analyses with the objective to find correlations between soil suppressiveness and biotic or abiotic soil characteristics. Significant differences in soil suppressiveness were found between the fields for all three diseases. Multiple regression indicated a significant correlation between suppressiveness against Rhizoctonia and the number of antagonistic Lysobacter spp., as well as with % active fungi and bacterial diversity. Grass-clover stimulated Rhizoctonia suppression as well as the presence of antagonistic Lysobacter spp. (mainly L. antibioticus and L. gummosus) in clay soils. Streptomyces suppression correlated with the number of antagonistic Streptomyces spp., % of active fungi and bacterial population size. The presence of antagonistic Streptomyces spp. correlated with a high fungal/bacterial biomass ratio. Verticillium suppression was only measured in 2004 and 2005, due to the inconsistent suppressiveness along the years. Nevertheless, a significant correlation with pH, potential nitrogen mineralization and bacterial biomass was found. Bacterial and fungal PCR-denaturing gel electrophoresis fingerprinting of bacterial and fungal communities, in general, did not significantly correlate with disease suppression. Highly significant explanatory factors of the composition of the dominating bacterial and fungal populations were % lutum, pH, C/N quotient, biomass and growth rate of bacteria. Additionally, the % of organic matter and years of organic farming were explaining significantly the composition of the bacterial population. Thus, significant correlations between several soil characteristics and suppressiveness of different soil-borne pathogens were found. For two of the three pathogens, suppression correlated with biotic soil characteristics combined with the presence of specific bacterial antagonists. Probably the soil suppressiveness measured in the organic fields is a combined effect of general and specific disease suppression.

The molecular fingerprinting technique (PCR-DGGE) of microbial populations was successfully adopted for soilless culture systems. Relatively low concentrations of micro-organisms in the nutrient solutions could be assessed. PCR-DGGE was applied in several experiments to compare the composition and diversity of the bacterial populations in different treatments, as well as at different locations and ages of the crop. Although numbers of culturable aerobic bacteria were similar in different rockwool treatments, the PCR-DGGE patterns showed that the bacterial composition and diversity differed. The bacterial composition in treatments which were suppressive to Pythium aphanidermatum (i.e. previously used rockwool and used rockwool that was recolonised after autoclaving) differed from that in autoclaved used rockwool with a high disease incidence. Additionally, a shift in the bacterial populations during plant growth was seen. Samples of the nutrient solution from different locations in a closed soilless culture system, i.e. drain, effluent, and solution in the rockwool slabs, showed different banding patterns. The results summarised in this paper showed that PCR-DGGE is a powerful tool to study the microflora under different conditions.

Non-pathogenic soilborne microorganisms can promote plant growth, as well as suppress diseases. Plant growth promotion is taken to result from improved nutrient acquisition or hormonal stimulation. Disease suppression can occur through microbial antagonism or induction of resistance in the plant. Several rhizobacterial strains have been shown to act as plant growth-promoting bacteria through both stimulation of growth and induced systemic resistance (ISR), but it is not clear in how far both mechanisms are connected. Induced resistance is manifested as a reduction of the number of diseased plants or in disease severity upon subsequent infection by a pathogen. Such reduced disease susceptibility can be local or systemic, result from developmental or environmental factors and depend on multiple mechanisms. The spectrum of diseases to which PGPR-elicited ISR confers enhanced resistance overlaps partly with that of pathogen-induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced basal resistance of the plant that depends on the signalling compounds jasmonic acid and salicylic acid, respectively, and pathogens are differentially sensitive to the resistances activated by each of these signalling pathways. Root-colonizing Pseudomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors. Conversely, plants can alter root exudation and secrete compounds that interfere with quorum sensing (QS) regulation in the bacteria. Such two-way signalling resembles the interaction of root-nodulating Rhizobia with legumes and between mycorrhizal fungi and roots of the majority of plant species. Although ISR-eliciting rhizobacteria can induce typical early defence-related responses in cell suspensions, in plants they do not necessarily activate defence-related gene expression. Instead, they appear to act through priming of effective resistance mechanisms, as reflected by earlier and stronger defence reactions once infection occurs.