The microbial changes during the biological control of cucumber damping-off disease using biocontrol agents and reductive soil disinfestation

Cucumber damping-off disease mainly caused by Rhizoctonia solani leads to serious loss in agricultural production. In this study, the effective and environmentally friendly methods, using two biological agents combined with applying organic matter or reductive soil disinfestation (RSD), were performed to control the disease. Real-time PCR and MiSeq pyrosequencing were used to investigate the microbial community changes during the biocontrol process. The results showed that the applications of organic matter and antagonists (Ant) significantly decreased R. solani population and disease incidence, and increased soil microbial population and activity. However, antagonists application combined with RSD (RSD + Ant) behaved much better in nearly all aspects, and facilitated to maintain the population and activity of antagonists. Compared to the applications of organic matter and antagonists, the combination of RSD and antagonists changed soil microbial community structure to a larger extent. In conclusion, the applications of biocontrol agents and organic matter improved soil microbial community and decreased cucumber damping-off disease incidence, but the combination of RSD and the antagonists was a more promising method for the improvement of soil microbial community and the stable and successful control of the disease.     

Solid-state fermentation of agro-industrial wastes to produce bioorganic fertilizer for the biocontrol of Fusarium wilt of cucumber in continuously cropped soil

Agro-industrial wastes of cattle dung, vinegar-production residue and rice straw were solid-state fermented by inoculation with Trichoderma harzianum SQR-T037 (SQR-T037) for production of bioorganic fertilizers containing SQR-T037 and 6-pentyl-α-pyrone (6PAP) to control Fusarium wilt of cucumber in a continuously cropped soil. Fermentation days, temperature, inoculum and vinegar-production residue demonstrated significant effects on the SQR-T037 biomass and the yield of 6PAP, based on fractional factorial design. Three optimum conditions for producing the maximum SQR-T037 biomass and 6PAP yield were predicted by central composite design and validated. Bioorganic fertilizer containing 8.46 log₁₀ ITS copies g⁻¹ dry weight of SQR-T037 and 1291.73 mg kg⁻¹ dry weight of 6PAP, and having the highest (p < 0.05) biocontrol efficacy, was achieved at 36.7 fermentation days, 25.9 °C temperature, 7.6% inoculum content, 41.0% vinegar-production residue, 20.0% rice straw and 39.0% cattle dung. This is a way to offer a high value-added use for agro-industrial wastes.     

Pectinolytic activity of bacteria isolated from soil and two fungal strains during submerged fermentation

Seven different strains were selected for their ability to degrade citrus pectin. Alkaline pectinases were produced by five bacterial soil isolates, whereas two fungal strains produced pectinase in an acidic environment. The bacteria were isolated from soil of a plum orchard in Northern Ireland. These isolates produced significant amounts of pectin lyase (PL) and polygalacturonase (PG) with maximum activities of 30.1 and 29.1 U/ml respectively. Fungal strains Aspergillus sp. and PN-1 produced four different pectinolytic activities; endo-PG, exo-PG, pectin esterase (PE) and PL. The Aspergillus sp. produced higher amounts of pectinase than PN-1. The Aspergillus sp. excreted highly stable pectinases, which may be of importance for industrial applications.     

Suppression of tomato bacterial wilt by anaerobic soil disinfestation and associations with production of antagonistic compounds

Plant and Soil - Anaerobic soil disinfestation (ASD) has been proven to be an effective and environmentally friendly method for controlling soil-borne plant diseases. Mechanisms of ASD-mediated...     

Production of fumonisins by Fusarium moniliforme strains isolated from corn grain

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).     

Production of fungal cell wall degrading enzymes by a biocontrol strain of Bacillus subtilis AF 1

Fungal cell wall degrading chitinases and glucanases attained significance in agriculture, medicine, and environment management. The present study was conducted to describe the optimum conditions required for the production of beta-1,4-N-acetyl glucosaminidase (NAGase) and beta-1,3-glucanase by a biocontrol strain of Bacillus subtilis AF 1. B. subtilis AF 1 was grown in minimal medium with colloidal chitin (3.0%) and yeast extract (0.3% YE ) and incubated at pH 7.0 and 30 degrees C on constant shaker at 180 rpm for 6 days produced highest amounts of NAGase. Presence of 0.5 mM of phenyl methyl sulfonyl fluoride (PMSF) and 0.04% of Tween 20 further improved the enzyme production. B. subtilis AF 1 grown in minimal medium with laminarin (1%) and yeast extract (0.3%) for 3 days produced maximum amount of beta-1,3-glucanase. These conditions can be further scaled-up for large-scale production of NAGase and beta-1,3-glucanase by B. subtilis AF 1.     

Potential of soil-derived fungal biocontrol agents applied as a soil amendment and a seed coating to control Verticillium wilt of sugar beet

Verticillium dahliae (Vd) is an emerging threat to sugar beet production. Control measures such as fungicides are not available and the utilisation of resistant cultivars is very limited. Hence, we explored the potential of two soil-derived fungal biocontrol agents (BCAs), Fusarium oxysporum F2 (FoF2) and Verticillium tricorpus 1808 (Vt1808), against Verticillium wilt of sugar beet. Pathogenicity tests revealed that Vd caused over 90% disease incidence and severity and led to a significant yield reduction, whereas BCAs neither inhibited nor promoted plant growth. Germination rate was higher in BCA-treated seeds compared to untreated ones. Viability of both BCAs was significantly reduced after six months of storage in liquid methylcellulose formulation (MC), while BCA concentrations remained stable on stored seeds treated with MC. In contrast, Vt1808 produced in sand-rye flour formulation remained 100% viable after storage. Similarly, post-storage analysis of the FoF2 talcum powder formulation revealed improved colony forming units, but increments were not significant. In vitro, both BCAs caused no growth inhibition zones and only insignificant Vd growth reductions were observed. In the greenhouse, soil amendment with a higher dose of FoF2:Vt1808 mixture resulted in substantial reductions of disease severity on the crown (33.3%), disease incidence (55.6%) and disease severity (68.8%) on the beet as well as an improved yield (32.9%). In contrast, seed coating did not reduce symptoms on the crown and on the beet. In the field, both BCAs did not provide significant disease reductions. Nevertheless, a promising result was achieved with the application of FoF2. Despite the need for improvement of the biocontrol activity, the results of this study demonstrate the suitability of the optimised BCA formulations for utilisation in commercial sugar beet production.     

Solution conformation and dynamics of a fungal cell wall polysaccharide isolated from Microsporum gypseum

The conformational and dynamical features of a branched mannan isolated from a fungal cell wall have been analysed by homo and heteronuclear NMR methods, employing different magnetic fields. 1HNMR cross relaxation times have been obtained for this polysaccharide and have been interpreted qualitatively using different motional models. 13C NMR relaxation parameters (T1, T2, NOE) have also been measured and interpreted using different approximations based on the Lipari and Szabo model free approach. The analysis of the data indicate the existence of important flexibility for the different linkages of the polysaccharide. Motions in the range of 4–6 ns contribute to the relaxation of the macromolecule, although faster internal motions in the 500 ps and 100 ps timescales are also present. These time scales indicate that segmental motions as well as internal motions around the glycosidic linkages are the major sources of relaxation for this molecule at 318 K. Molecular dynamics simulations have also been performed. The obtained results also indicate that the polysaccharide possess a substantial amount of conformational freedom.     

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml⁻¹. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml⁻¹). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani. This revised version was published online in November 2006 with corrections to the Cover Date.     

Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals

Thirty-four isolates of the eight most common Fusarium species isolated from Norwegian cereals; F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. sporotrichioides, F. torulosum and F. tricinctum were studied for their cytotoxicity and ability to produce mycotoxins. The strains were cultivated on rice, and analysed for trichothecenes (all species), zearalenone (all species), fusarochromanone (F. equiseti), wortmannin (F. torulosum), moniliformin and enniatins (F. avenaceum, F. tricinctum and F. torulosum). The cytotoxicity of the extracts were examined with an (in vitro) MTT-cell culture assay. All F. graminearum and five of seven F. culmorum isolates belonged to chemotype IA, producing deoxynivalenol and 3-acetyl-deoxynivalenol, while the two other F. culmorum strains were nivalenol producers (chemotype II). The F. equiseti isolates and one of the F. poae isolates produced both type A and B trichothecenes, and relatively large quantities of fusarochromanone were detected in the F. equiseti cultures. All Fusarium species studied showed significant cytotoxicity, but with a large variation between species, and also within each species. F. sporotrichioides and F. equiseti showed the highest average cytotoxicity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579

Fusarium oxysporum f. sp. cucumerinum is a destructive pathogen on cucumber (Cucumis sativus L.) seedlings and the causal organism of crown and root rot of cucumber plants. An isolate of B579, which was identified as Bacillus subtilis by 16S rDNA sequences analysis, was selected from 158 bacteria isolates as the best antagonist against F. oxysporum by dual plate assay. The production of chitinase, β-1, 3-glucanase, siderophores, indole-3-acetic acid (IAA), hydrogen cyanide (HCN), and phosphate solubilization, by B579 were screened with the selected medium by in vitro tests. The cell-free culture filtrate of B579, with a concentration of 20% (v/v), could result in the vacuolation, swelling and lysis of hyphae. Besides, it could blacken, shrunk and hindered the germination of conidia of F. oxysporum at the concentration of ≥80% (v/v). When applied as inoculants, B579 (10⁸ c.f.u. ml^?1) was able to reduce disease incidence by 73.60%, and promote seedling growth in pot trial studies. The activities of plant defense-related enzyme, peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were significantly increased in plants treated with B579. Interestingly, a higher content of IAA, an important plant growth regulator, was detected in B579 treated plants. Furthermore, seed-soaking with B579 exhibited a better biological control effect (Biocontrol effect 73.60%) and plant growth promoting ability (Vigor Index 4,177.53) than root-irrigation (50.88% and 3,575.77, respectively), suggesting the potential use of B579 as a seed-coating agent.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Degradation of reactive dyes by ozonation and oxalic acid-assimilating bacteria isolated from soil

Ozonation and treatment of wastewaters with oxalic acid-assimilating bacterium was attempted for the complete degradation of reactive dyes. Oxalic acid-assimilating bacterium, Pandoraea sp. strain EBR-01, was newly isolated from soil under bamboo grove and was identified to be a member of the genus Pandoraea by physicochemical and biochemical tests including 16S rDNA sequence analysis. The bacterium was grown optimally at pH 7 and temperature of 30 degrees C under the laboratory conditions. Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Black 5 (RB5) and Remazol Brilliant Blue R (RBBR) were used in degradation experiments. At the initial reactive dye concentrations of 500 mg/l and the ozonation time of 80 min, it was confirmed that 75-90 mg/l oxalic acid was generated from reactive dyes by ozonation. Microbial treatment using EBR-01 greatly decreased the amount of oxalic acid in the mixture after 48 h, but it was not removed completely. TOC/TOC(0) of reactive dye solutions was also decreased to 80-90% and 20-40% by ozonation and microbial treatment using EBR-01, respectively. The study confirmed that consecutive treatments by ozone and microorganisms are efficient methods to mineralize reactive dyes.     

The fungal cell wall: Modern concepts of its composition and biological function

This review deals with the cell wall (CW), a poorly known surface structure of the cell of mycelial fungi. Data are presented concerning (i) isolation techniques and purity control methods securing the absence of the cytoplasm content in the CW fraction and (ii) the chemical composition of the CW. The structural (backbone) and intrastructural components of the CW, such as aminopolysaccharides, α- and β-glucans, proteins, lipids, uronic acids, hydrophobins, sporopollenin, and melanins, are discussed in detail. Special attention is given to chitin and its novel function in terms of protecting the cells from stress as well as to the differences of this fungal aminopolysaccharide from the chitin of algae and Arthropoda. The apical growth of hyphae and the involvement of special microvesicles in morphogenesis of a fungal cell are discussed. Data on the enzymes involved in CW synthesis and lysis are presented. In conclusion, the functional role of the fungal CW is discussed in juxtaposition to the surface structures of higher eukaryotes.     

Biochemical studies on the cell wall degradation of Fusarium oxysporum f.sp. lycopersici race 2 by lytic enzymes from Mucorales for its biocontrol

The lysis of cell wall, cell wall fractions (F1S—alkali and water soluble, F1I—alkali soluble and water insoluble, and F4—β-chitin-glucan) and chlamydospores of Fusarium oxysporum f.sp. lycopersici race 2 (Fol 2) with crude enzyme preparations from Mucorales (Absidia californica, Mortierella vinacea, Mucor hiemalis, Phycomyces blakesleeanus, Rhizopus stolonifer and Syncephalastrum racemosum ) has been studied during an incubation period of 72 h. The lysis of the F1S fraction and chlamydospores was followed by the release of reducing substances. The lysis of the other fractions and cell walls was followed by percentage of decrease in optical density (lysis degree) and release of reducing substances. A correlation between the degradation of cell walls and cell wall fractions was observed. The soluble fraction was the most resistant to lysis by these enzymes, with the highest release of reducing substances at 4% of the material as glucose equivalent using the crude enzyme preparation from Mucor hiemalis. Crude enzyme preparations from Rhizopus stolonifer produced the highest lysis (33.4%) and release of reducing substances (22.1%) from the Fol 2 cell wall. The enzymes from M. hiemalis produced the highest degradation of chlamydospores. A possible biological control of Fol 2 with lytic enzymes from R. stolonifer or M. hiemalis is indicated.
We wish to thank Dr J. Tello for the Fol 2 strain, Dr P. Melgarejo for her valuable discussion and Mrs Carmelina Rodríguez for her technical assistance. This work was supported by the Spanish programmes 'Plan Nacional de Investigación Científica y Desarrollo Tecnológico' (AGR91-0014-C02-02) and 'Plan Nacional de Investigación de la Comunidad de Madrid' (C052/91) and DGICYT PB92–0001. We also wish to thank Norel S.A. for their contract to one of us (O.M. Nuero).     

Contamination of pine nuts by fumonisin produced by strains of Fusarium proliferatum isolated from Pinus pinea

AIMS: To test the ability of Fusarium proliferatum strains isolated from Pinus pinea to synthesize fumonisin B(1) (FB(1)) in pine nuts. METHOD AND RESULTS: Eleven strains were inoculated in moist sterile shelled pine nuts and whole pine nuts, and incubated for 28 days. Moist sterile maize was inoculated in parallel as an optimum substrate for FB(1) production by Fusarium species. CONCLUSIONS: Six of the strains produced FB(1) in shelled pine nuts, and two of them did so in whole pine nuts. IMPACT AND SIGNIFICANCE OF THE STUDY: F. proliferatum is able to grow and produce FB(1) in the husk of pine nuts and the mycotoxin can be found in the nut once shelled. Consequently, FB(1) may be an important hazard in pine nuts.     

Recognition of the fungal cell wall by innate immune receptors

Human cells have a variety of receptors that innately recognize conserved structures on the fungal cell wall. Major receptors include dectin-1, which recognizes β1,3-glucans; mannose receptors, which recognize mannans, and Toll-like receptors 2 and 4. The fungal cell wall is a potent activator of complement, which results in deposition of fragments of the third component of complement that serve as ligands for complement receptors. The nature of the innate immune response is dictated by the relative amount each of these receptors is stimulated. Innate recognition can lead to destruction of the invading fungus and/or initiation of an adaptive immune response. Fungi have a variety of strategies to avoid innate recognition, including masking of ligands and changing their surface properties by phase transition.