Phenylacetic acid-producing Rhizoctonia solani represses the biosynthesis of nematicidal compounds in vitro and influences biocontrol of Meloidogyne incognita in tomato by Pseudomonas fluorescens strain CHA0 and its GM derivatives

AIMS: The aim of the present investigation was to determine the influence of Rhizoctonia solani and its pathogenicity factor on the production of nematicidal agent(s) by Pseudomonas fluorescens strain CHA0 and its GM derivatives in vitro and nematode biocontrol potential by bacterial inoculants in tomato. METHODS AND RESULTS: One (Rs7) of the nine R. solani isolates from infected tomato roots inhibited seedling emergence and caused root rot in tomato. Thin layer chromatography revealed that culture filtrates of two isolates (Rs3 and Rs7) produced brown spots at Rf-values closely similar to synthetic phenylacetic acid (PAA), a phytotoxic factor. Filtrates from isolate Rs7, amended with the growth medium of P. fluorescens, markedly repressed nematicidal activity and PhlA'-'LacZ reporter gene expression of the bacteria in vitro. On the contrary, isolate Rs4 enhanced nematicidal potential of a 2,4-diacetylphloroglucinol overproducing mutant, CHA0/pME3424, of P. fluorescens strain CHA0 in vitro. Therefore, R. solani isolates Rs4 and Rs7 were tested more rigorously for their potential to influence biocontrol effectiveness of the bacterial agents. Methanol extract of the culture filtrates of PAA-producing isolate Rs7 resulting from medium amended with phenylalanine enhanced fungal repression of the production of nematicidal agents by bacteria, while amendments with zinc or molybdenum eliminated such fungal repression, thereby restoring bacterial potential to cause nematode mortality in vitro. A pot experiment was carried out, 3-week-old tomato seedlings were infested with R. solani isolates Rs4 or Rs7 and/or inoculated with Meloidogyne incognita, the root-knot nematode. The infested soil was treated with aqueous cell suspensions (10(8) CFU) of P. fluorescens strain CHA0 or its GM derivatives or left untreated (as a control). Observations taken 45 days after nematode inoculation revealed that, irrespective of the bacterial treatments, galling intensity per gram of fresh tomato roots was markedly higher in soil amended with isolate Rs4 than in Rs7-amended soils. Soil amendments with R. solani and the bacterial antagonists resulted in substantial reductions of the number of galls per gram of root. These results are contradictory to those obtained under in vitro conditions where culture filtrates of PAA-positive Rs7 repressed the production of nematicidal compounds. Plants grown in Rs7-amended soils, with or without bacterial inoculants, had lesser shoot and root weights than plants grown in nonamended or Rs4-amended soils. Moreover, amendments with Rs7 substantially retarded root growth and produced necrotic lesions that reduced the number of entry sites for invasion and subsequent infection by nematodes. Populations of P. fluorescens in the tomato rhizosphere were markedly higher in Rs7-amended soils. CONCLUSIONS: PAA-producing virulent R. solani drastically affects the potential of P. fluorescens to cause death of M. incognita juveniles in vitro and influences bacterial effectiveness to suppress nematodes in tomato roots. SIGNIFICANCE AND IMPACT OF THE STUDY: As most agricultural soils are infested with root-infecting fungi, including R. solani, it is likely that some PAA-producing isolates of the fungus may also be isolated from such soils. The inhibitory effect of PAA-producing R. solani on the biosynthesis of nematicidal agent(s) critical in biocontrol may reduce or even eliminate the effectiveness of fluorescent pseudomonads against root-knot nematodes, both in nursery beds and in field conditions. Introduction of bacterial inoculants, for the control of any plant pathogen, should be avoided in soils infested with PAA-producing R. solani. Alternatively, the agents could be applied together with an appropriate quantity of fungicide or chemicals such as zinc to create an environment more favourable for bacterial biocontrol action.     

Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.     

Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots

Extracellular polysaccharides play an important role in the formation of bacterial biofilms. We tested the biofilm-forming ability of two mutant strains with increased production of acidic extracellular polysaccharides compared with the wild-type biocontrol strain Pseudomonas fluorescens CHA0. The anchoring of bacteria to axenic nonmycorrhizal and mycorrhizal roots as well as on extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices was investigated. The nonmucoid wild-type strain P. fluorescens CHA0 adhered very little on all surfaces, whereas both mucoid strains formed a dense and patchy bacterial layer on the roots and fungal structures. Increased adhesive properties of plant-growth-promoting bacteria may lead to more stable interactions in mixed inocula and the rhizosphere.     

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.     

Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities

Pseudomonas fluorescens strain CHA0, a root colonizing bacterium, has a broad spectrum of biocontrol activity against plant diseases. However, strain CHA0 is unable to utilize 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of plant ethylene, as a sole source of nitrogen. This suggests that CHA0 does not contain the enzyme ACC deaminase, which cleaves ACC to ammonia and alpha-ketobutyrate, and was previously shown to promote root elongation of plant seedlings treated with bacteria containing this enzyme. An ACC deaminase gene, together with its regulatory region, was transferred into P. fluorescens strains CHA0 and CHA96, a global regulatory gacA mutant of CHA0. ACC deaminase activity was expressed in both CHA0 and CHA96. Transformed strains with ACC deaminase activity increased root length of canola plants under gnotobiotic conditions, whereas strains without this activity had no effect. Introduction of ACC deaminase genes into strain CHA0 improved its ability to protect cucumber against Pythium damping-off, and potato tubers against Erwinia soft rot in small hermetically sealed containers. In contrast, ACC deaminase activity had no significant effect on the ability of CHA0 to protect tomato against Fusarium crown and root rot, and potato tubers against soft rot in large hermetically sealed containers. These results suggest that (i) ACC deaminase activity may have lowered the level of plant ethylene thereby increasing root length; (ii) the role of stress-generated plant ethylene in susceptibility or resistance depends on the host-pathogen system, and on the experimental conditions used; and (iii) the constructed strains could be developed as biosensors for the role of ethylene in plant diseases.     

Low-level herbivory by root-knot nematodes (Meloidogyne incognita) modifies root hair morphology and rhizodeposition in host plants (Hordeum vulgare)

Low amounts of root infestation by plant parasitic nematodes are suggested to increase nutrient supply and in turn enhance microbial activity and net mineralization rate in the rhizosphere. These effects are generally related to “leakage” of plant-derived metabolites from damaged roots. Besides leakage, the present study examines other nematode–host interactions such as alterations in root exudation and morphology, which were almost not considered yet. This includes undamaged root parts in order to assess systemic plant response. The root-knot nematode Meloidogyne incognita (Kofoid and White 1919; Chitwood 1949) and barley (Hordeum vulgare L. cv. Europa) was used as model system. Host plants were grown in mini-rhizotrons inoculated with 0, 2,000, 4,000 or 8,000 M. incognita for 4 weeks. Root morphology, rhizodeposition (sugars, carboxylates, amino acids), and rhizosphere microbial communities (PLFAs) were assessed. In treatments with 4,000 nematodes, shoot biomass, total N and P content increased by the end of the experiment. Generally, an enhanced release of plant metabolites (sugars, carboxylates, amino acids) from the apical root zone occurred 1 week after inoculation with 4,000 and 8,000 M. incognita, indicating root leakage. Low levels of root herbivory stimulated root hair elongation in both infected and uninfected roots. These systemic changes in root morphology likely contributed to the increased sugar exudation in uninfected roots in all nematode treatments at 3 weeks after inoculation. Root-knots formed a separate microhabitat within the root-system. They were characterised by decreased rhizodeposition and increased fungal to bacterial ratio in the adhering rhizosphere soil. The present study provides the first evidence that, apart from leakage, nematode root herbivory at background levels induces local and systemic effects on root morphology and exudation, which in turn may affect plant performance.     

Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean

AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean. METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated. Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles. The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not. Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils. In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased. Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues. CONCLUSIONS: Soil inoculation with Ps. fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning. Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g. antibiotic-overproducing Pseudomonas strains) released into the environment.     

Plant Species, Host Age and Host Genotype Effects on Meloidogyne incognita Biocontrol by Pseudomonas fluorescens Strain CHA0 and its Genetically-Modified Derivatives

Pseudomonas fluorescens strain CHA0 and its antibiotic overproducing derivative CHA0/pME3424 repeatedly reduced Meloidogyne incognita galling on tomato, brinjal, mungbean and soya bean roots but not in chilli. An antibiotic‐deficient derivative, CHA89, did not reduce nematode invasion in any of the plant species tested. When plant species were compared, bacterial inoculants afforded better protection to tomato, mungbean and soya bean roots against root‐knot nematodes than to brinjal and chilli. Antibiotic overproducing strain CHA0/pME3424 markedly reduced fresh shoot weights of chilli and mungbean while antibiotic‐deficient strain CHA89 enhanced fresh shoot weights of mungbean. While strains CHA0 had no significant impact on fresh root weights of any of the plant species, strain CHA0/pME3424 consistently reduced fresh root weights of brinjal and mungbean. In none of the plant species the bacterial strains had an influence on protein contents of the leaves. Regardless of the plant species, the three bacterial strains did not differ markedly in their rhizosphere colonization pattern. However, colonization was highest in brinjal rhizosphere and lowest in the mungbean rhizosphere. A slight host genotype effect on the biocontrol performance of the bacterial inoculants was also detected at cultivar level. When five soya bean cultivars were compared, biocontrol bacteria exhibited best suppression of the root‐knot nematode in cv. Ajmeri. Antibiotic overproducing strain CHA0/pME3424 substantially reduced fresh shoot weights of the soya bean cultivars Centuray 84 and NARC‐I while strain CHA89 enhanced shoot weights of the cultivars Ajmeri, William‐82 and NARC‐II. Wild type strain CHA0 had no significant impact on fresh shoot weights of any of the soya bean cultivars. Strain CHA0/pME3424 reduced fresh weights of root of Century 84, NARC‐I and NARC‐II while strain CHA89 increased root weights. Bacterial rhizosphere colonization was highest in variety NARC‐I and lowest in variety Ajmeri. Plant age had a significant impact on the biocontrol performance of bacterial inoculants against nematodes. The biocontrol effect of all bacterial strains was more prominent during early growth stage (7 days after nematode inoculation). A strong negative correlation between bacterial rhizosphere colonization and nematode invasion in soya bean roots was observed.     

Role of ethylene in the protection of tomato plants against soil-borne fungal pathogens conferred by an endophytic Fusarium solani strain

An endophytic fungal isolate (Fs-K), identified as a Fusarium solani strain, was obtained from root tissues of tomato plants grown on a compost which suppressed soil and foliar pathogens. Strain Fs-K was able to colonize root tissues and subsequently protect plants against the root pathogen Fusarium oxysporum f.sp. radicis-lycopersici (FORL), and elicit induced systemic resistance against the tomato foliar pathogen Septoria lycopersici. Interestingly, attenuated expression of certain pathogenesis-related genes, i.e. PR5 and PR7, was detected in tomato roots inoculated with strain Fs-K compared with non-inoculated plants. The expression pattern of PR genes was either not affected or aberrant in leaves. A genetic approach, using mutant tomato plant lines, was used to determine the role of ethylene and jasmonic acid in the plant's response to infection by the soil-borne pathogen F. oxysporum f.sp. radicis-lycopersici (FORL), in the presence or absence of isolate Fs-K. Mutant tomato lines Never ripe (Nr) and epinastic (epi1), both impaired in ethylene-mediated plant responses, inoculated with FORL are not protected by isolate Fs-K, indicating that the ethylene signalling pathway is required for the mode of action used by the endophyte to confer resistance. On the contrary, def1 mutants, affected in jasmonate biosynthesis, show reduced susceptibility to FORL, in the presence Fs-K, which suggests that jasmonic acid is not essential for the mediation of biocontrol activity of isolate Fs-K.     

Systemic Resistance in Tomato Induced by Biocontrol Bacteria Against the Root-Knot Nematode, Meloidogyne javanica is Independent of Salicylic Acid Production

Salicylic acid (SA)‐mediated induction of systemic resistance by Pseudomonas aeruginosa strain 7NSK2 and P. fluorescens strain CHA0 against soil‐borne fungi and viruses have been reported. The role of SA biosynthesis in the enhancement of defence mechanism against plant‐parasitic nematodes by these bacterial strains in tomato is not known. To better understand the importance of SA in rhizobacteria‐mediated suppression of root‐knot nematodes, biocontrol potential of SA‐negative or SA‐overproducing mutants against Meloidogyne javanica was evaluated with their respective wild type counter parts. Culture supernatant of 7NSK2, CHA0 and their respective mutants caused significant mortality of M. javanica juveniles in vitro. SA deletion in 7NSK2 and SA overproduction in CHA0 did not influence bacterial efficacy to cause nematode deaths. Similarly, culture supernatants resulting from King's B liquid medium amended with FeCl₃ did not influence nematicidal activity of the bacterial strains. Strain CHA0 induced juvenile deaths more than 7NSK2 did. In pot experiments, the bacterial strains applied in unsterilized sandy loam soil markedly reduced final nematode population densities in roots and subsequent root‐knot infection in tomato seedlings. SA‐negative or overproducing derivatives prevented tomato roots in kinetics similar to those with their respective wild types. When soil iron concentration was lowered by the addition of ethylenediamine di(o‐hydroxyphenylacetic acid), nematode biocontrol by the bacterial strains (both wild type and mutants) remained unaltered. To understand the mechanism involved in rhizobacteria‐mediated suppression of root‐knot nematode in tomato, bacterial performance was assessed in a split root trial in which one‐half of the root system was treated with bacterium while the other inoculated with nematode. Compared with the controls, application of the bacterial cell suspension to one‐half of the root system lowered the populations of root‐knot nematode in non‐bacterized nematode‐treated sections indicating enhanced defence in the non‐bacterized half. With respect to nematode infection, mutants induced systemic resistance to a similar extent as that caused by the wild types in both wild type tomato and NahG tomato plants. It is concluded that fluorescent pseudomonads induce systemic resistance against root‐knot nematode via a signal transduction pathway, which is independent of SA accumulation in roots.     

Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P.   f luorescens i nsecticidal t oxin) that is related to the insect toxin Mcf ( M akes c aterpillars f loppy) of the entomopathogen Photorhabdus luminescens , a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella . In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria.     

A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Infected grains are often contaminated with mycotoxins harmful to humans and animals. During the past decade, F. graminearum has caused several severe epidemics of head scab in wheat and barley. In order to understand molecular mechanisms regulating fungal development and pathogenicity in this pathogen, we isolated and characterized a MAP kinase gene, MGV1, which is highly homologous to the MPS1 gene in Magnaporthe grisea. The MGV1 gene was dispensable for conidiation in F. graminearum but essential for female fertility during sexual reproduction. Vegetative growth of mgv1 deletion mutants was normal in liquid media but reduced on solid media. Mycelia of the mgv1 mutants had weak cell walls and were hypersensitive to cell wall degrading enzymes. Interestingly, the mgv1 mutants were self-incompatible when tested for heterokaryon formation, and their virulence was substantially reduced. The ability of the mutants to accumulate trichothecene mycotoxins on inoculated wheat was also greatly reduced. Our data suggest that MGV1 in F. graminearum is involved in multiple developmental processes related to sexual reproduction, plant infection, and cell wall integrity.     

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.     

A soil-free root observation system for the study of root-microorganism interactions in maize

Background and aims

The root surface of a plant usually exceeds the leaf area and is constantly exposed to a variety of soil-borne microorganisms. Root pathogens and pests, as well as belowground interactions with beneficial microbes, can significantly influence a plants' performance. Unfortunately, the analysis of these interactions is often limited because of the arduous task of accessing roots growing in soil. Here, we present a soil-free root observation system (SF-ROBS) designed to grow maize (Zea mays) plants and to study root interactions with either beneficial or pathogenic microbes.

Methods

The SF-ROBS consists of pouches lined with wet filter paper supplying nutrient solution.

Results

The aspect of maize grown in the SF-ROBS was similar to soil-grown maize; the plant growth was similar for the shoot but different for the roots (biomass and length increased in the SF-ROBS). SF-ROBS-grown roots were successfully inoculated with the hemi-biotrophic maize fungal pathogen Colletotrichum graminicola and the beneficial rhizobacteria Pseudomonas putida KT2440. Thus, the SF-ROBS is a system suitable to study two major belowground phenomena, namely root fungal defense reactions and interactions of roots with beneficial soil-borne bacteria.

Conclusions

This system contributes to a better understanding of belowground plant microbe interactions in maize and most likely also in other crops.     

Sex-Related Responses of Populus cathayana Shoots and Roots to AM Fungi and Drought Stress

We investigated the impact of drought and arbuscular mycorrhizal (AM) fungi on the morphological structure and physiological function of shoots and roots of male and female seedlings of the dioecious plant Populus cathayana Rehder. Pot-grown seedlings were subjected to well watered or water-limiting conditions (drought) and were grown in soil that was either inoculated or not inoculated with the AM fungus Rhizophagus intraradices. No significant differences were found in the infection rates between the two sexes. Drought decreased root and shoot growth, biomass and root morphological characteristics, whereas superoxide radical (O₂–) and hydrogen peroxide content, peroxidase (POD) activity, malondialdehyde (MDA) concentration and proline content were significantly enhanced in both sexes. Male plants that formed an AM fungal symbiosis showed a significant increase in shoot and root morphological growth, increased proline content of leaves and roots, and increased POD activity in roots under both watering regimes; however, MDA concentration in the roots decreased. By contrast, AM fungi either had no effect or a slight negative effect on the shoot and root growth of female plants, with lower root biomass, total biomass and root/shoot ration under drought. In females, MDA concentration increased in leaves and roots under both watering regimes, and the proline content and POD activity of roots increased under drought conditions; however, POD activity significantly decreased under well-watered conditions. These findings suggest that AM fungi enhanced the tolerance of male plants to drought by improving shoot and root growth, biomass and the antioxidant system. Further investigation is needed to unravel the complex effects of AM fungi on the growth and antioxidant system of female plants.     

Effects of the endophyte Epichloë coenophiala on the root microbial community and growth performance of tall fescue in different saline-alkali soils

Soil salinization is detrimental to plant growth and yield in agroecosystems worldwide. Epichloë endophytes, a class of clavicipitaceous fungi, enhance the resistance of host plants to saline-alkali stress. This study explored the effects of the systemic fungal endophyte Epichloë coenophiala on the root microbial community and growth performance of tall fescue (Lolium arundinaceum) growing under different saline-alkali stress conditions. Structural equation modeling (SEM) was conducted to analyze the direct and indirect effects (mediated by root microbial community diversity and soil properties) of the endophyte on the growth of tall fescue under saline-alkali stress. The endophyte-infected plants produced higher shoot and root biomass compared to endophyte-free plants under saline-alkali stress (200 and 400 mM). Endophyte infection increased the fungal community diversity and altered its composition in the roots, decreasing the relative abundance of Ascomycota and increasing that of Glomeromycota. Furthermore, endophyte infection decreased the bacterial community diversity and the relative abundance of dominant Proteobacteria. SEM showed that endophyte infection increased the shoot and root biomass under saline-alkali stress (200 and 400 mM) by increasing the arbuscular mycorrhizal fungal diversity in the roots, and soil total nitrogen and phosphorus concentrations. Therefore, it is important to examine aboveground microbes as factors influencing plant growth in saline-alkali stress by affecting belowground microbes and soil chemical properties.     

Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.     

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.