Metabolic Profiling of Endophytic Bacteria in Relation to Their Potential Application as Components of Multi-Task Biopreparations.

Agricultural crops are exposed to various abiotic and biotic stresses that can constrain crop productivity. Focusing on a limited subset of key groups of organisms has the potential to facilitate the monitoring of the functions of human-managed ecosystems. Endophytic bacteria can enhance plant stress resistance and can help plants to cope with the negative impacts of stress factors through the induction of different mechanisms, influencing plant biochemistry and physiology. In this study, we characterise endophytic bacteria isolated from different plants based on their metabolic activity and ability to synthesise 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD), the activity of hydrolytic exoenzymes, the total phenolic compounds (TPC) and iron-complexing compounds (ICC). Test GEN III MicroPlate indicated that the evaluated endophytes are highly metabolically active, and the best used substrates were amino acids, which may be important in selecting potential carrier components for bacteria in biopreparations. The ACCD activity of strain ES2 (Stenotrophomonas maltophilia) was the highest, whereas that of strain ZR5 (Delftia acidovorans) was the lowest. Overall, the obtained results indicated that ∼91.3% of the isolates were capable of producing at least one of the four hydrolytic enzymes. In addition, most of the tested strains produced ICC and TPC, which play a significant role in reducing stress in plants. The results of this study suggest that the tested endophytic bacterial strains can potentially be used to mitigate climate change-associated stresses in plants and to inhibit plant pathogens.

Differences in the Production of Extracellular Polymeric Substances (EPS) and Other Metabolites of Plenodomus (Leptosphaeria) Infecting Winter Oilseed Rape (Brassica napus L.).

Species of the genus Plenodomus (Leptosphaeria) are phytopathogens of the Brassicaceae family, which includes oilseed rape. The spores of these fungi spread by airborne transmission, infect plants, and cause crop losses. The secondary metabolism of P. lingam and P. biglobosus was studied and compared, with the main focus being on the ability to produce Extracellular Polymeric Substances (EPS). In spite of the 1.5-2-fold faster growth rate of P. biglobosus on Czapek-Dox and other screening media, the average yield of EPS in this fungus was only 0.29 g/L, compared to that of P. lingam (0.43 g/L). In turn, P. biglobosus showed a higher capacity to synthesise IAA, i.e., 14 µg/mL, in contrast to <1.5 µg/mL produced by P. lingam. On the other hand, the P. lingam strains showed higher ß-glucanase activity (350-400 mU/mL), compared to 50-100 mU/mL in P. biglobosus. Invertase levels were similar in both species (250 mU/mL). The positive correlation between invertase activity and EPS yield contrasted with the absence of a correlation of EPS with ß-glucanase. Plenodomus neither solubilised phosphate nor used proteins from milk. All strains showed the ability to synthesise siderophores on CAS agar. P. biglobosus exhibited the highest efficiency of amylolytic and cellulolytic activity.

Trichoderma: The Current Status of Its Application in Agriculture for the Biocontrol of Fungal Phytopathogens and Stimulation of Plant Growth.

Rhizosphere filamentous fungi of the genus Trichoderma, a dominant component of various soil ecosystem mycobiomes, are characterized by the ability to colonize plant roots. Detailed knowledge of the properties of Trichoderma, including metabolic activity and the type of interaction with plants and other microorganisms, can ensure its effective use in agriculture. The growing interest in the application of Trichoderma results from their direct and indirect biocontrol potential against a wide range of soil phytopathogens. They act through various complex mechanisms, such as mycoparasitism, the degradation of pathogen cell walls, competition for nutrients and space, and induction of plant resistance. With the constant exposure of plants to a variety of pathogens, especially filamentous fungi, and the increased resistance of pathogens to chemical pesticides, the main challenge is to develop biological protection alternatives. Among non-pathogenic microorganisms, Trichoderma seems to be the best candidate for use in green technologies due to its wide biofertilization and biostimulatory potential. Most of the species from the genus Trichoderma belong to the plant growth-promoting fungi that produce phytohormones and the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme. In the present review, the current status of Trichoderma is gathered, which is especially relevant in plant growth stimulation and the biocontrol of fungal phytopathogens.

Differences in Production, Composition, and Antioxidant Activities of Exopolymeric Substances (EPS) Obtained from Cultures of Endophytic Fusarium culmorum Strains with Different Effects on Cereals.

Exopolymeric substances (EPS) can determine plant-microorganism interactions and have great potential as bioactive compounds. The different amounts of EPS obtained from cultures of three endophytic Fusarium culmorum strains with different aggressiveness-growth promoting (PGPF), deleterious (DRMO), and pathogenic towards cereal plants-depended on growth conditions. The EPS concentrations (under optimized culture conditions) were the lowest (0.2 g/L) in the PGPF, about three times higher in the DRMO, and five times higher in the pathogen culture. The EPS of these strains differed in the content of proteins, phenolic components, total sugars, glycosidic linkages, and sugar composition (glucose, mannose, galactose, and smaller quantities of arabinose, galactosamine, and glucosamine). The pathogen EPS exhibited the highest total sugar and mannose concentration. FTIR analysis confirmed the ß configuration of the sugars. The EPS differed in the number and weight of polysaccharidic subfractions. The EPS of PGPF and DRMO had two subfractions and the pathogen EPS exhibited a subfraction with the lowest weight (5 kDa). The three EPS preparations (ethanol-precipitated EP, crude C, and proteolysed P) had antioxidant activity (particularly high for the EP-EPS soluble in high concentrations). The EP-EPS of the PGPF strain had the highest antioxidant activity, most likely associated with the highest content of phenolic compounds in this EPS.

A Report on Fungal (1→3)-α-d-glucans: Properties, Functions and Application.

The cell walls of fungi are composed of glycoproteins, chitin, and α- and ß-glucans. Although there are many reports on ß-glucans, α-glucan polysaccharides are not yet fully understood. This review characterizes the physicochemical properties and functions of (1→3)-α-d-glucans. Particular attention has been paid to practical application and the effect of glucans in various respects, taking into account unfavourable effects and potential use. The role of α-glucans in plant infection has been proven, and collected facts have confirmed the characteristics of Aspergillus fumigatus infection associated with the presence of glucan in fungal cell wall. Like ß-glucans, there are now evidence that α-glucans can also stimulate the immune system. Moreover, α-d-glucans have the ability to induce mutanases and can thus decompose plaque.

Endophytic Bacteria Potentially Promote Plant Growth by Synthesizing Different Metabolites and their Phenotypic/Physiological Profiles in the Biolog GEN III MicroPlateTM Test.

Endophytic bacteria, as the most promising components of effective, biofertilizers biostimulating and biocontrol preparations, should be very intensively obtained from various plants and studied in terms of the conditions determining the potential ability to promote plant growth. For this reason, endophytic bacteria have been isolated from both stems and roots of up to six systematically distant species of vascular plants: one species belonging to the seedless vascular plants (Monilophyta), and five seed plants (Spermatophyta). The 23 isolated strains represented nine genera: Delftia, Stenotrophomonas, Rhizobium, Brevundimonas, Variovorax, Achromobacter, Novosphingobium, Comamonas and Collimonas, notably which were closely related-belonging to the phylum Proteobacteria. Stenotrophomonas sp. strains showed the greatest ability to synthesize indole-3-acetic acid (IAA)-like compounds, while Achromobacter sp. strains produced the highest levels of siderophores. The presence of the nifH gene and nitrogen binding activity was demonstrated for 95% of the strains tested. Stenotrophomonas maltophila (ES2 strain) showed the highest metabolic activity based on Biolog GEN III test. The ability to solubilize phosphate was determined only for three tested strains from genus: Delftia, Rhizobium and Novosphingobium. The presented work demonstrated that the metabolic and phenotypic properties of plant growth-promoting endophytes are correlated with the genus of bacteria and are not correlated with the host plant species or part of plant (stem, root).

Phytohormones (Auxin, Gibberellin) and ACC Deaminase In Vitro Synthesized by the Mycoparasitic Trichoderma DEMTkZ3A0 Strain and Changes in the Level of Auxin and Plant Resistance Markers in Wheat Seedlings Inoculated with this Strain Conidia.

Both hormonal balance and plant growth may be shaped by microorganisms synthesizing phytohormones, regulating its synthesis in the plant and inducing plant resistance by releasing elicitors from cell walls (CW) by degrading enzymes (CWDE). It was shown that the Trichoderma DEMTkZ3A0 strain, isolated from a healthy rye rhizosphere, colonized the rhizoplane of wheat seedlings and root border cells (RBC) and caused approximately 40% increase of stem weight. The strain inhibited (in over 90%) the growth of polyphagous Fusarium spp. (F. culmorum, F. oxysporum, F. graminearum) phytopathogens through a mechanism of mycoparasitism. Chitinolytic and glucanolytic activity, strongly stimulated by CW of F. culmorum in the DEMTkZ3A0 liquid culture, is most likely responsible for the lysis of hyphae and macroconidia of phytopathogenic Fusarium spp. as well as the release of plant resistance elicitors. In DEMTkZ3A0 inoculated plants, an increase in the activity of the six tested plant resistance markers and a decrease in the concentration of indoleacetic acid (IAA) auxin were noted. IAA and gibberellic acid (GA) but also the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) enzyme regulating ethylene production by plant were synthesized by DEMTkZ3A0 in the liquid culture. IAA synthesis was dependent on tryptophan and negatively correlated with temperature, whereas GA synthesis was positively correlated with the biomass and temperature.

Antifungal Properties of Fucus vesiculosus L. Supercritical Fluid Extract Against Fusarium culmorum and Fusarium oxysporum.

In this study, potential antifungal properties of a brown alga Fucus vesiculosus were evaluated. The algal extract was obtained with the use of supercritical fluid extraction (scCO2) at a temperature of 50 °C under a pressure of 300 bar. The aqueous solution of the extract at the concentration of 0.05%, 0.2%, 0.5% and 1.0% was studied against pathogenic fungi on a liquid RB medium. This study is the first report on antifungal properties of the brown algae F. vesiculosus scCO2 extract against Fusarium culmorum and Fusarium oxysporum phytopathogens. The concentrations of the studied extract (0.5% and 1.0%) were demonstrated to have an ability to inhibit 100% growth of macroconidia within 144 h, as well as an ability to cause their total degradation. As a result of the study, the antifungal effect of fucosterol against F. culmorum was also indicated. The total macroconidia growth was inhibited by 1.0% fucosterol. Moreover, at lower concentrations (0.05-0.2%) of fucosterol, macroconidia were characterized by shorter length and structural degradation was observed. The mycelial growth of Fusarium oxysporum (Fo38) by 1% scCO2 F. vesiculosus extract was analyzed at the level of 48% after 168 h of incubation, whereas 100% extract was found to be effective in F. culmorum (CBS122) and F. oxysporum (Fo38) growth inhibition by 72% and 75%, respectively after 168 h of incubation.

Bacterial Isolate Inhabiting Spitsbergen Soil Modifies the Physiological Response of Phaseolus coccineus in Control Conditions and under Exogenous Application of Methyl Jasmonate and Copper Excess.

The aim of the study was to demonstrate the potential of the promotion and regulation of plant physiology and growth under control and copper stress conditions, and the impact of the exogenous application of methyl jasmonate on this potential. Runner bean plants were treated with methyl jasmonate (1 or 10 µM) (J; J1 or J10) and Cu (50 µM), and inoculated with a bacterial isolate (S17) originating from Spitsbergen soil, and identified as Pseudomonas luteola using the analytical profile index (API) test. Above- and under-ground plant parts were analyzed. The growth parameters; the concentration of the photosynthetic pigments, elements, flavonoids (FLAVO), phenolics (TPC), allantoin (ALLA), and low molecular weight organic acids (LMWOAs); the activity of antioxidant enzymes and enzymes of resistance induction pathways (e.g., superoxide dismutase (SOD), catalase (CAT), ascorbate (APX) and guaiacol (GPX) peroxidase, glucanase (GLU), and phenylalanine (PAL) and tyrosine ammonia-lyase (TAL)), and the antioxidant capacity (AC) were studied. The leaves exhibited substantially higher ALLA and LMWOA concentrations as well as PAL and TAL activities, whereas the roots mostly had higher activities for a majority of the enzymes tested (i.e., SOD, CAT, APX, GPX, and GLU). The inoculation with S17 mitigated the effect of the Cu stress. Under the Cu stress and in the presence of J10, isolate S17 caused an elevation of the shoot fresh weight, K concentration, and TAL activity in the leaves, and APX and GPX (also at J1) activities in the roots. In the absence of Cu, isolate S17 increased the root length and the shoot-to-root ratio, but without statistical significance. In these conditions, S17 contributed to a 236% and 34% enhancement of P and Mn, respectively, in the roots, and a 19% rise of N in the leaves. Under the Cu stress, S17 caused a significant increase in FLAVO and TPC in the leaves. Similarly, the levels of FLAVO, TPC, and AC were enhanced after inoculation with Cu and J1. Regardless of the presence of J, inoculation at Cu excess caused a reduction of SOD and CAT activities, and an elevation of GPX. The effects of inoculation were associated with the application of Cu and J, which modified plant response mainly in a concentration-dependent manner (e.g., PAL, TAL, and LMWOA levels). The conducted studies demonstrated the potential for isolate S17 in the promotion of plant growth.

Physiological Diversity of Spitsbergen Soil Microbial Communities Suggests Their Potential as Plant Growth-Promoting Bacteria.

The objective of the study was to assess the physiological diversity and metabolic activity of the soil bacterial communities inhabiting Spitsbergen soils in search of bacterial abilities facilitating plant growth promotion. In the soil, the total number of culturable microorganisms, the number of their individual physiological groups (including Siderophore Synthesizing; SSB and Phosphate Solubilizing Bacteria; PSB), the dehydrogenase (DH) activity, and the ability to utilize sources of C, N, P (EcoPlate) were analysed. In bacterial isolates, siderophores production, ACC (1-aminocyclopropane-1-carboxylate) deaminase (ACCD) activity, IAA (indole-3-acetic acid) synthesis were examined. The isolates were applied to the seeds of Phaseolus coccineus regarding their germination and root length. The results showed differences between copio- and oligotrophic bacteria. A usually high number of SSB was accompanied by the raised number of PSB. A bigger number of SSB was connected with low values of Fe in the soil. High DH activity was assisted by greater number of copio- and oligotrophic bacteria, raised average well color development value, and N and C contents in the soil. Germination index was more alike relative seed germination than relative root growth. IAA concentration and ACCD activity were conversely related. Synthesis of siderophores was matched with ACCD activity and its high level was combined with elevated germination index. In spite of different localization of soil samples, some isolates proved similar traits of activity. Distinct affiliation of isolates and their various localizations were displayed. Among all isolates tested, some possessed one main trait of activity, but most of them had two or more significant features for potential plant growth stimulation. These isolates could be an important source of useful bacteria.

Synthesis of Indoleacetic Acid, Gibberellic Acid and ACC-Deaminase by Mortierella Strains Promote Winter Wheat Seedlings Growth under Different Conditions.

The endogenous pool of phytoregulators in plant tissues supplied with microbial secondary metabolites may be crucial for the development of winter wheat seedlings during cool springs. The phytohormones may be synthesized by psychrotrophic microorganisms in lower temperatures occurring in a temperate climate. Two fungal isolates from the Spitzbergen soils after the microscopic observations and "the internal transcribed spacer" (ITS) region molecular characterization were identified as Mortierella antarctica (MA DEM7) and Mortierella verticillata (MV DEM32). In order to study the synthesis of indoleacetic acid (IAA) and gibberellic acid (GA), Mortierella strains were grown on media supplemented with precursor of phytohormones tryptophan at 9, 15 °C, and 20 °C for nine days. The highest amount of IAA synthesis was identified in MV DEM32 nine-day-culture at 15 °C with 1.5 mM of tryptophan. At the same temperature (15 °C), the significant promoting effect (about 40% root and shoot fresh weight) of this strain on seedlings was observed. However, only MA DEM-7 had the ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with the highest efficiency at 9 °C and synthesized IAA without tryptophan. Moreover, at the same conditions, the strain was confirmed to possess the strong promoting effect (about 40% root and 24% shoot fresh weight) on seedlings. Both strains synthesized GA in all tested terms and temperatures. The studied Mortierella strains had some important traits that led them to be considered as microbial biofertilizers components, improving plant growth in difficult temperate climates.

Identification and characteristics of biological agents in work environment of medical emergency services in selected ambulances.

OBJECTIVES: Assessment of microbial air quality and surface contamination in ambulances and administration offices as a control place without occupational exposure to biological agents; based on quantitative and qualitative analysis of bacteria, yeasts and filamentous fungi found in collected samples. MATERIAL AND METHODS: The sampling was done by wet cyclone technology using the Coriolis recon apparatus, imprint and swab methods, respectively. In total, 280 samples from 28 ambulances and 10 offices in Warszawa were tested. Data was analyzed using Shapiro-Wilk normality test, Kruskal-Wallis test with α = 0.05. P value ≤ 0.05 was considered as significant. RESULTS: The levels of air contamination were from 0 to 2.3×101 colony-forming unit (CFU)/m3 for bacteria and for yeast and filamentous fungi were from 0 to 1.8×101 CFU/m3. The assessment of office space air samples has shown the following numbers of microorganisms: bacteria from 3.0×101 to 4.2×101 CFU/m3 and yeast and filamentous fungi from 0 to 1.9×101 CFU/m3. For surface contamination the mean bacterial count in ambulances has been between 1.0×101 and 1.3×102 CFU/25 cm2 and in offices - between 1.1×101 and 8.5×101 CFU/25 cm2. Mean fungal count has reached the level from 2.8×100 to 4.2×101 CFU/25 cm2 in ambulances and 1.3×101 to 5.8×101 CFU/25 cm2 in offices. The qualitative analysis has revealed the presence of Acinetobacter spp. (surfaces), coagulase - negative Staphylococci (air and surfaces), Aspergillus and Penicillium genera (air and surfaces). CONCLUSIONS: The study has revealed a satisfactory microbiological quantity of analyzed air and surface samples in both study and control environments. However, the presence of potentially pathogenic microorganisms in the air and on surfaces in ambulances may endanger the medical emergency staff and patients with infection. Disinfection and cleaning techniques therefore should be constantly developed and implemented. Int J Occup Med Environ Health 2017;30(4):617-627.

Mobilization of cadmium from Festuca ovina roots and its simultaneous immobilization by soil in a root-soil-extractant system (in vitro test).

Mobilization of cadmium accumulated in Festuca ovina L. roots and simultaneous immobilization of this metal by soil were studied in three chambers connected into one system containing: (1) roots in an extractant solution, (2) soil in an extractant solution, and (3) extractant solution alone. Six extractants sterilized by filtration were used: 0.1 M NaNO3 (NA), 0.1 mM desferrioxamine B (NA + DFOB) and 1 mM citric acid (NA + CA) in NA, and a water extract of soil (SE) supplemented with the same compounds. SE mobilized 53% of the Cd introduced to the system with roots. The addition of DFOB or CA to SE increased Cd extraction from roots by 17%, while the same compounds introduced to NA did not change mobilization of Cd (60% efficiency). Regardless of the extractant used, mobilization of Cd from roots was about 25% lower when extraction was done in a control system without soil. The metal released from roots was gradually immobilized by the soils loaded into all systems during a 4-day incubation. Sequential extractions of Cd from the soils showed that the metal released from roots with NA was stabilized mainly by soil Mn and Fe oxides, while that released with SE was stabilized by soil organic matter.

Negative correlation between mycological surfaces pollution in hospital emergency departments and blood monocytes phagocytosis of healthcare workers.

The aim of the present study was to find a possible relationship between the presence of yeast and filamentous fungi in hospital emergency departments and the activity levels of blood granulocytes and monocytes in emergency personnel. The study of mycological pollution was conducted in winter; the samples were collected from 10 Warsaw hospitals emergency departments (HE D) and in 10 control locations (office spaces) and included air samples and swabbing of floor and walls. The blood for immunological investigation was taken in spring, from 40 men, 26 to 53 years old, healthcare workers of these departments, who have been working for at least 5 years in their current positions, and from 36 corresponding controls, working in control offices. Evaluation of blood leukocyte subpopulations was done by hematological analyzer and cytometry analysis and monocyte and granulocyte phagocytosis by Phagotest. There were no significant differences in the level of mycological contamination between the test and control places. The qualitative analysis of the surfaces and air samples revealed a prevalence of strains belonging to Aspergillus spp. and Penicillium spp. genus. Statistical analysis revealed the existence of negative correlation between the number of phagocytizing blood monocytes and fungi spores content on floor and wall surfaces in hospital emergency departments (r = -0.3282, p < 0.05 and positive correlation between the number of phagocytizing monocytes in the blood of office workers and fungi pollution of control offices (r = 0.4421, p < 0.01).

Extracellular polysaccharides from Ascomycota and Basidiomycota: production conditions, biochemical characteristics, and biological properties.

Fungal polysaccharides (PSs) are the subject of research in many fields of science and industry. Many properties of PSs have already been confirmed and the list of postulated functions continues to grow. Fungal PSs are classified into different groups according to systematic affinity, structure (linear and branched), sugar composition (homo- and heteropolysaccharides), type of bonds between the monomers (ß-(1 → 3), ß-(1 → 6), and α-(1 → 3)) and their location in the cell (cell wall PSs, exoPSs, and endoPSs). Exopolysaccharides (EPSs) are most frequently studied fungal PSs but their definition, classification, and origin are still not clear and should be explained. Ascomycota and Basidiomycota fungi producing EPS have different ecological positions (saprotrophic and endophytic, pathogenic or symbiotic-mycorrhizae fungi); therefore, EPSs play different biological functions, for example in the protection against environmental stress factors and in interactions with other organisms. EPSs obtained from Ascomycota and Basidiomycota fungal cultures are known for their antioxidant, immunostimulating, antitumor, and antimicrobial properties. The major objective of the presented review article was to provide a detailed description of the state-of-the-art knowledge of the effectiveness of EPS production by filamentous and yeast Ascomycota and Basidiomycota fungi and techniques of derivation of EPSs, their biochemical characteristics, and biological properties allowing comprehensive analysis as well as indication of similarities and differences between these fungal groups. Understanding the role of EPSs in a variety of processes and their application in food or pharmaceutical industries requires improvement of the techniques of their derivation, purification, and characterization. The detailed analyses of data concerning the derivation and application of Ascomycota and Basidiomycota EPSs can facilitate development and trace the direction of application of these EPSs in different branches of industry, agriculture, and medicine.

Characterization of callase (ß-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

Hydrolysis of fungal and plant cell walls by enzymatic complexes from cultures of Fusarium isolates with different aggressiveness to rye (Secale cereale).

The efficiency of hydrolysis of fungal (Fusarium spp.) cell wall and rye root cell wall by crude enzymatic complexes from (42-day-old) cultures of three F. culmorum isolates, a plant growth-promoting rhizosphere isolate (PGPF) DEMFc2, a deleterious rhizosphere isolate (DRMO) DEMFc5, and a pathogenic isolate DEMFc37, as well as two other, pathogenic isolates belonging to F. oxysporum and F. graminearum species was studied. In the enzymatic complexes originating from the Fusarium spp. cultures, the activities of the following cell wall-degrading enzymes were identified: glucanases, chitinases, xylanases, endocellulases, exocellulases, pectinases, and polygalacturonases. The preparation originating from a culture of the PGPF isolate was the least efficient in plant cell wall (PCW) hydrolysis. There were no significant differences in the efficiency of PCW hydrolysis between preparations from cultures of the DRMO and the pathogenic isolates. PGPF was the most efficient in liberating reducing sugars and N-acetylglucosamine (GlcNAc) from fungal cell walls (FCW). Xylanase activities of the enzymatic complexes were strongly positively (R > +0.9) correlated with their efficiency in hydrolyzing PCW, whereas chitinase activities were correlated with the efficiency in FCW hydrolysis.

Activities of cell wall degrading enzymes in autolyzing cultures of three Fusarium culmorum isolates: growth-promoting, deleterious and pathogenic to rye (Secale cereale).

Release of cell wall degrading enzymes, CWDE, (glucanases, chitinases, xylanases, endocellulases, exocellulases, pectinases and polygalacturonases) was compared for three Fusarium culmorum isolates, two nonpathogenic rhizosphere isolates (a plant growth promoting [PGPF] and a deleterious [DRMO]) and one root pathogen, grown on media supplemented with one of these C sources: glucose, chitin, plant (rye root) and fungal (Fusarium) cell wall. The degree of autolysis determined after 42 d in the medium containing glucose was 15% for PGPF and DRMO and 20% for pathogenic isolate. The organic compounds added to the growth medium differentially affected the activity of the individual enzymes released by the particular isolates. The activities of xylanases and endocellulases released to the plant cell wall-amended medium by the PGPF isolate were significantly lower than the activities of these enzymes released by the DRMO and the pathogenic isolates. The activity of pectinases was repressed by glucose. The activities of acidic hydrolases were greater than those of alkaline hydrolases. Principal component analysis revealed that the activities of the CWDE found in the supernatants of the autolyzing F. culmorum cultures could be clustered into two distinct groups. One group included pectinase, exocellulase and polygalacturonase and all the remaining tested hydrolases in the other, suggesting that enzymes from either group might act in synergy during cell wall degradation. The differences in the activities of the individual CWDE released to the culture by the particular isolates are considered to be one of the key factors responsible for the observed types of plant-fungal interactions.

Interactions between rye (Secale cereale) root border cells (RBCs) and pathogenic and nonpathogenic rhizosphere strains of Fusarium culmorum.

Interactions of rye (Secale cereale) root border cells (RBCs), generated during plant growth and surrounding the root cap, with nonpathogenic rhizosphere Fusarium culmorum isolates: DEMFc2 (PGPF) and DEMFc5 (DRMO), and a pathogenic strain DEMFc37 were studied in test tube experiments. The effect of water-suspended RBCs released from the rye root cap on the rate of macroconidia germination and hyphae (mycelial) growth of F. culmorum strains was also examined. It was found that root caps of 3-d-old rye seedlings (with the root length of 20mm) were surrounded with a layer of RBCs generated in a number specific for this plant species of 1980+/-30. Introduction of the macroconidia of the tested F. culmorum strains into the root zone of 3-d-old seedlings resulted, after 3d of incubation, in the formation of mantle-like structures only in the rhizosphere of plants inoculated with the pathogenic DEMFc37 strain. The macroconidia were suspended in (1) water, (2) a water mixture with root caps deprived of RBCs, (3) Martin medium, (4) malt extract broth, and (5) a water mixture with rye RBCs, and their percentage germination was determined during 96-h incubation at 20 degrees C. Germination of the macroconidia of all the tested F. culmorum strains suspended in the rich growth media (Martin and malt extract broth) and in the mixture with RBCs was significantly speeded up. While only an average of 16.6 % of macroconidia suspended in water germinated after 96-h incubation, more than 90 % of those suspended in the growth media or in the mixture with RBCs germinated after 24h of incubation. In all the treatments, the highest rate of macroconidia germination was found in suspensions of the pathogenic strain and the lowest in macroconidial suspensions of the PGPF strain. The stimulatory effect of RBCs was not specific to the pathogenic strain. Nevertheless, microscopic observation revealed that it was only in the suspension containing a mixture of rye RBCs and macroconidia of the pathogenic strain that after 48-h incubation compact clusters of hyphae and RBCs, resembling mantle-like structures found in the root zone of plants inoculated only with the pathogenic strain but not inoculated with DRMO and PGPF strain, were formed.

Multiple copies of rosR and pssA genes enhance exopolysaccharide production, symbiotic competitiveness and clover nodulation in Rhizobium leguminosarum bv. trifolii.

Rhizobium leguminosarum bv. trifolii exopolysaccharide (EPS) plays an important role in determining symbiotic competence. The pssA gene encoding the first glucosyl-IP-transferase and rosR encoding a positive transcriptional regulator are key genes involved in the biosynthesis and regulation of EPS production. Mutation in pssA resulted in deficiency in EPS production and rosR mutation substantially decreased the amount of EPS. Both mutants induced nodules but the bacteria were unable to fix nitrogen. Defective functions of pssA and rosR mutants were fully restored by wild type copies of the respective genes. Introduction of multiple rosR and pssA gene copies on the plasmid vector pBBR1MCS-2 into five R. leguminosarum bv. trifolii nodule isolates resulted in significantly increased growth rates, EPS production and the number of nodules on clover roots. Increase in fresh and dry shoot mass of clovers and nodule occupation was also statistically significant. Interestingly, additional copies of pssA but particularly rosR gene, increased strains' competitiveness in relation to the wild type parental strains nearly twofold. Overall, experimental evidence is provided that increased amount of EPS beneficially affects R. leguminosarum bv. trifolii competitiveness and symbiosis with clover.