Functional characterization of a novel hydrocarbonoclastic Pseudomonas sp. strain PUP6 with plant-growth-promoting traits and antifungal potential

A novel hydrocarbonoclastic bacterium was isolated from rice rhizospheric soil using an enrichment culture technique. Detailed taxonomic studies identified the organism, designated strain PUP6, as a member of the genus Pseudomonas. The bacterium grew in minimal medium amended with n-alkane members of hydrocarbons, n-dodecane (C12H26), n-hexadecane (C16H34), n-octadecane (C18H38), n-octacosane (C28H58); and petroleum fractions such as crude oil and lubricating oil when provided as sole carbon and energy source. Degradation of these n-alkane hydrocarbons and oils in minimal salts medium by strain PUP6 was estimated using gas chromatography with a flame ionization detector. In addition to its hydrocarbonoclastic properties, this bacterium exhibits a broad spectrum of fungal antibiosis against various phytopathogenic fungi. An antifungal metabolite produced by strain PUP6 was isolated, characterized and identified as phenazine-1-carboxamide on the basis of nuclear magnetic resonance and mass spectroscopic analyses. Strain PUP6 also produced plant-growth-promoting siderophores, indoleacetic acid (IAA), phosphate solubilizing enzymes, and fungal cell wall degrading enzymes such as protease and chitinase. This study can be considered as the first report on n-alkane hydrocarbon and oil degradation by a rhizosphere soil bacterium that exhibits biofertilizing and biocontrol traits. Due to its innate multiple functional traits beyond its role in degradation of hydrocarbons, strain PUP6 may be used as plant-growth-promoting rhizobacterium and biocontrol agent against phytopathogenic fungi.     

Lytic enzyme complex of an antagonistic Bacillus sp. X-b: isolation and purification of components

Bacillus sp. X-b, a biocontrol agent against certain plant pathogenic fungi, secretes a complex of hydrolytic enzymes, composed of chitinase, chitosanase, laminarinase, lipase and protease. Homogenized mycelium of basidiomycete Macrolepiota procera induced activities of these enzymes more effectively than colloidal chitin or partially purified cell walls of another basidiomycete Polyporus squamosus. Subjected to a multi-step purification, the specific activity of chitinase increased 36-fold, chitosanase 69-fold, lipase 44-fold and laminarinase 15-fold. Partially purified chitinase showed two major bands with molecular masses of 46,000 and 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while chitosanase and lipase appeared as single bands with molecular masses of 27,000 and 62,000, respectively.     

The Tunisian oasis ecosystem is a source of antagonistic Bacillus spp. producing diverse antifungal lipopeptides

The use of microbial products has become a promising alternative approach to controlling plant diseases caused by phytopathogenic fungi. Bacteria isolated from the date palm tree rhizosphere of the Tunisian oasis ecosystem could provide new biocontrol microorganisms adapted to extreme conditions, such as drought, salinity and high temperature. The aim of this study was to screen bacteria isolated from the rhizosphere of the date palm tree for their ability to inhibit phytopathogenic fungi, and to identify molecules responsible for their antifungal activity. Screening for antifungal activity was performed on twenty-eight isolates. Five antagonistic isolates were selected and identified as different species of Bacillus using phenotypical methods and a molecular approach. The five antagonistic Bacillus isolated showed tolerance to abiotic stresses (high temperature, salinity, drought). Their ability to produce lipopeptides was investigated using a combination of two techniques: PCR amplification and MALDI-ToF mass spectrometry. Analyses revealed that the antagonistic isolates produced a high diversity of lipopeptides that belonged to surfactin, fengycin, iturin and kurstakin families. Their antagonistic activity, related to their capacity for producing diverse antifungal lipopeptides and their tolerance to abiotic stresses, highlighted Bacillus strains isolated from the rhizosphere of the date palm tree as potential biocontrol agents for combatting plant diseases in extreme environments.     

Biocontrol potential of Bacillus subtilis RH5 against sheath blight of rice caused by Rhizoctonia solani

The sheath blight disease of rice caused by Rhizoctonia solani is widely prevalent and one of the most destructive diseases, affecting rice cultivation and loss worldwide. In the present study, a set of twenty Bacillus isolates from saline soil of Uttar Pradesh were tested for their biocontrol activity against R. solani with the aim to obtain a potential strain for the control of sheath blight disease toward ecofriendly and sustainable agriculture. The results of dual-culture assay and scanning electron microscopic studies showed that the strain RH5 exhibited significant antagonistic activity (84.41%) against the fungal pathogen R. solani. On the basis of 16S rDNA sequencing analysis, the potential biocontrol strain RH5 was identified as Bacillus subtilis. Furthermore, the strain RH5 was characterized by different plant growth-promoting (PGP) activities and induction of defense-related enzymes in rice plants against R. solani. The strain RH5 posses various PGP attributes (indole acetic acid, siderophore, hydrogen cyanide production and phosphate, Zn, K solubility), hydrolytic enzymatic (chitinase, protease, cellulase, xylanase) activity, and presence of antimicrobial peptide biosynthetic genes (bacylisin, surfactin, and fengycin), which support the strain for efficient colonization of hyphae and its inhibition. Finally, the results of the greenhouse study confirmed that strain RH5 significantly increased plant growth and triggered resistance in rice plants through the production of defense-related antioxidant enzymes.     

Statistical optimization of medium components for production of extracellular chitinase by Basidiobolus ranarum: a novel biocontrol agent against plant pathogenic fungi

The influence of concentration of medium components such as colloidal chitin, lactose, malt extract, yeast extract, and peptone on the chitinase production from Basidiobolous ranarum at the flask level were studied by using statistical tool Central Composite Design (CCD) and analysed by Response Surface Methodology (RSM). The results revealed that colloidal chitin, malt extract and peptone had significant effect (P < 0.01) on the chitinase production at their individual levels. The polynomial equation of the model developed incorporates 3 linear, 3 quadratic and 5 interactive terms. Maximum chitinase production of 3.47 U ml^–1 was achieved with 1.5% colloidal chitin, 0.125% lactose, 0.025% malt extract and 0.075% peptone. After optimization, chitinase activity was increased by 7.71 fold. A second order polynomial equation was found to be useful for the development of efficient bioprocess for chitinase production. To screen the biotechnological potential of this enzyme, degradation of fungal mycelia by ammonium sulphate precipitate of the same was studied for several pathogenic fungi‐in vitro which showed promising results particularly against Rhizoctonia solani and Fusarium solani. This study provides the first evidence showing the effectiveness of RSM for the development of a robust statistical model for the chitinase production by Basidiobolus and for its application in the biocontrol of phytopathogenic fungi. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Antimicrobial properties of a non-toxic glycoprotein (WSG) from Withania somnifera (Ashwagandha)

A monomeric glycoprotein with a molecular mass of 28 kDa in SDS-PAGE was isolated from the Withania somnifera root tubers. The protein designated WSG (Withania somnifera glycoprotein) demonstrated potent antimicrobial activity against the phytopathogenic fungi and bacteria tested. Antifungal effect has been demonstrated in that WSG exerts a fungistastic effect by inhibiting spore germination and hyphal growth in the tested fungi. WSG showed potent antifungal activity against Aspergillus flavus, Fusarium oxysporum, F. verticilloides and antibacterial activity against Clvibacter michiganensis subsp. michiganensis. WSG is an acidic, non-toxic (trypsin-chymotrypsin) protease inhibitor. These results encourage further studies of WSG as a potential therapeutic agent for its antifungal activity.     

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients'' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.     

Anti-phytopathogen potential of endophytic actinobacteria isolated from tomato plants (Lycopersicon esculentum) in southern Brazil,and characterization of Streptomyces sp. R18(6), a potential biocontrol agent

Tomato plants (Lycopersicon esculentum) are highly susceptible to phytopathogen attack. The resulting intensive application of pesticides on tomato crops can affect the environment and health of humans and animals. The objective of this study was to select potential biocontrol agents among actinobacteria from tomato plants, in a search for alternative phytopathogen control. We evaluated 70 endophytic actinobacteria isolated from tomato plants in southern Brazil, testing their antimicrobial activity, siderophore production, indoleacetic acid production, and phosphate solubility. The actinomycete isolate with the highest antimicrobial potential was selected using the agar-well diffusion method, in order to optimize conditions for the production of compounds with antimicrobial activity. For this study, six growth media (starch casein-SC, ISP2, Bennett’s, Sahin, Czapek–Dox, and TSB), three temperatures (25 °C, 30 °C, and 35 °C) and different pH were tested. Of the actinobacteria tested, 88.6% showed antimicrobial activity against at least one phytopathogen, 72.1% showed a positive reaction for indoleacetic acid production, 86.8% produced siderophores and 16.2% showed a positive reaction for phosphate solubility. Isolate R18(6) was selected due to its antagonistic activity against all phytopathogenic microorganisms tested in this study. The best conditions for production were observed in the SC medium, at 30 °C and pH 7.0. The isolate R18(6) showed close biochemical and genetic similarity to Streptomyces pluricolorescens.     

Bio-control and plant growth promotion potential of siderophore producing endophytic Streptomyces from Azadirachta indica A. Juss

Three endophytic actinomycetes strains recovered from surface sterilized root tissues of Azadirachta indica A. Juss. (Meliaceae), plants were selected through tests for their potential as bio-control and plant growth promoting agents. It was also observed that the seed treated with the spore suspension of three selected strains of Streptomyces, significantly promoted plant growth and antagonized the growth of Alternaria alternata, causal agent of early blight disease in tomato plant. It was observed that the three selected strains prolifically produce IAA and siderophores that play vital role in promotion of plant growth and in suppression of Alternaria alternata. Interestingly, Streptomyces strain AzR-051 produced the highest amount of IAA at 13.73 μmol ml(-1) , compared to strains AzR-049 and AzR-010 9.22 μmol ml(-1) and 10.43 μmol ml(-1) respectively. It also produces siderophores higher than the other two strains. Thus these endophytic isolates have the potential as plant growth promoters as well as a bio-control agent, which is a useful trait for crop production in nutrient deficient soils.     

Selektion und biotechnische Herstellung von Kulturlösungen mikrobieller Antagonisten zur Unterdrückung phytopathogener Bodenpilze

Two strains of bacteria, Bacillus subtilis T 99 and Streptomyces sp. G 43 , antagonistic to phytopathogenic soil fungi, were isolated from natural substrates. In vitro tests indicated activities against different Fusarium species including formae speciales of Fusarium oxysporum, Stromatinia gladioli, Botrytis cinerea, Thanatephorus cucumeris, and other fungi. For both of the antagonists, biotechnical procedures were developed yielding culture broth for large-scale application. After dilution of these media antagonists were tested successfully in vivo by soil drenching treatments in a greenhouse carnation production facility. The application forms of the fermentation media developed were capable of suppressing wilt disease of Dianthus.     

Characterization of biocontrol bacterial strains isolated from a suppressiveness-induced soil after amendment with composted almond shells

The improvement in soil quality of avocado crops through organic amendments with composted almond shells has a positive effect on crop yield and plant health, and enhances soil suppressiveness against the phytopathogenic fungus Rosellinia necatrix. In previous studies, induced soil suppressiveness against this pathogen was related to stimulation of Gammaproteobacteria, especially some members of Pseudomonas spp. with biocontrol-related activities. In this work, we isolated bacteria from this suppressiveness-induced amended soil using a selective medium for Pseudomonas-like microorganisms. We characterized the obtained bacterial collection to aid in identification, including metabolic profiles, antagonistic responses, hybridization to biosynthetic genes of antifungal compounds, production of lytic exoenzymatic activities and plant growth-promotion-related traits, and sequenced and compared amplified 16S rDNA genes from representative bacteria. The final selection of representative strains mainly belonged to the genus Pseudomonas, but also included the genera Serratia and Stenotrophomonas. Their biocontrol-related activities were assayed using the experimental avocado model, and results showed that all selected strains protected the avocado roots against R. necatrix. This work confirmed the biocontrol activity of these Gammaproteobacteria-related members against R. necatrix following specific stimulation in a suppressiveness-induced soil after a composted almond shell application.     

Comparison of antifungal activities and 16S ribosomal DNA sequences of clinical and environmental isolates of Stenotrophomonas maltophilia

In recent years, the gram-negative bacterium Stenotrophomonas maltophilia has become increasingly important in biotechnology and as a nosocomial pathogen, giving rise to a need for new information about its taxonomy and epidemiology. To determine intraspecies diversity and whether strains can be distinguished based on the sources of their isolation, 50 S. maltophilia isolates from clinical and environmental sources, including strains of biotechnological interest, were investigated. The isolates were characterized by in vitro antagonism against pathogenic fungi and the production of antifungal metabolites and enzymes. Phenotypically the strains showed variability that did not correlate significantly with their sources of isolation. Clinical strains displayed remarkable activity against the human pathogenic fungus Candida albicans. Antifungal activity against plant pathogens was more common and generally more severe from the environmental isolates, although not exclusive to them. All isolates, clinical and environmental, produced a range of antifungal metabolites including antibiotics, siderophores, and the enzymes proteases and chitinases. From 16S ribosomal DNA sequencing analysis, the isolates could be separated into three clusters, two of which consisted of isolates originating from the environment, especially rhizosphere isolates, and one of which consisted of clinical and aquatic strains. In contrast to the results of other recent investigations, these strains could be grouped based on their sources of isolation, with the exception of three rhizosphere isolates. Because there was evidence of nucleotide signature positions within the sequences that are suitable for distinguishing among the clusters, the clusters could be defined as different genomovars of S. maltophilia. Key sequences on the 16S ribosomal DNA could be used to develop a diagnostic method that differentiates these genomovars.     

Genotypic and phenotypic diversity of Anabaena isolates from diverse rice agro-ecologies of India

The genus Anabaena is one of the commonly observed genera in the rice fields in South-east Asia. Diversity analyses of a set of 70 Anabaena strains (including 67 strains isolated from diverse rice agro ecologies of India, and three International Reference/Type strains), was carried out using morphological and molecular datasets. The pattern of growth in liquid and solid medium and microscopic observations revealed tremendous diversity in the Anabaena germplasm analysed. The species wise distribution in different soil types and soil pH revealed that Anabaena iyengarii was present at pH ranging from 5.5-8.5 and all the species of Anabaena except A. oscillarioides were present in alluvium soils. Molecular profiling using primers based on HipTG, STRR(mod) and STRR1A sequences generated specific fingerprints for individual isolates. STRR1A was observed to be the most informative and useful for differentiating the isolates. Analyses of a combined dataset, including both morphological and molecular data, proved highly effective in discerning the genetic relationships among the 70 Anabaena strains. The present study provided useful information for the development of a comprehensive database based on the distribution of Anabaena strains in diverse agro ecologies of India and identified useful primers for PCR based differentiation of isolates.     

Antifungal activity of aqueous and methanolic extracts from the Mediterranean sea cucumber,Holothuria polii

Crude extracts and semi-purified fractions (F5, F6) from the Mediterranean sea cucumber, Holothuria polii, collected from the bay of Tabarka (Tunisia coast) were evaluated for their antifungal activity against filamentous fungi and yeast. The activity was determined in vitro, using the well diffusion test in the casitone agar medium. Both the aqueous and the methanolic extracts were found to produce, in a concentration-related manner (600–1500 μg/well) a significant antifungal activity. The semi-purified fractions (F5, F6) of both extracts exhibited also a significant antifungal activity in a concentration-related manner (150–300 μg/well). The strains of Aspergillus fumigatus were more susceptible to these extracts and derived fractions, while those of Trichophyton rubrum were found to be less susceptible. No activity was observed against strains of Candida albicans. These findings suggest that the polar active fractions (F5, F6) obtained from aqueous and methanolic extracts could contain a new antifungal compound(s). The purification and the determination of chemical structure of compound(s) of these active fractions are under investigation.     

Detection of virulence factors and antifungal susceptibility of human and avian Aspergillus flavus isolates

Aspergillus flavus is the second leading cause of invasive and non-invasive aspergillosis. Secretion of hydrolytic enzymes is considered as a virulence factor in this species. Our work aimed to study in vitro production of some virulence factors, to evaluate the biofilm production against human and avian A. flavus isolates and to investigate the antifungal susceptibility agents. Hydrolytic enzymes, biofilm production and molecular typing were studied for 62 human and 36 avian A. flavus isolates by specific solid media and six microsatellite markers. The susceptibility to antifungal agents was evaluated for 37 human isolates. All human and avian A. flavus isolates showed positive activities of extracellular hydrolase: phospholipase, protease and hemolysin. A positive elastase activity was seen in 64.51% of human A. flavus isolates and 86.1% of avian A. flavus isolates. All A. flavus in these two populations formed biofilms. Statistical significant difference was observed for the mean phospholipase activities (P = 0.025) and biofilm quantification (P = 0.0001) between human and avian A. flavus isolates. The in vitro susceptibility results showed a resistance in 83.7%, 81.08% and 16.21% of A. flavus isolates respectively to amphotericin B, itraconazole and posaconazole. No association was noted between all virulence factors and the genotypes of human and avian isolates. Our study allowed us to show that human strains have a higher production of extracellular hydrolases and biofilm then avian strains. These virulence factors appear to act synergistically to contribute to the virulence of A. flavus strains. Moreover, significant correlation between virulence patterns and antifungal susceptibility profiles was observed.     

Regulation of two homodimer hexosaminidases in the mycoparasitic fungus Trichoderma asperellum by glucosamine

Trichoderma asperellum is a mycoparasitic fungus which is used as a biocontrol agent against plant pathogens. Its hydrolytic enzymes take part in its parasitic interaction, degrading the pathogen cell wall and thereby helping to control disease. One of those enzymes, -N-acetyl-d-glucosaminidase (GlcNAcase), degrades chitin, which is a major component of the cell wall of many plant-pathogenic fungi. Two GlcNAcases of T. asperellum T203, designated EXC1Y and EXC2Y, were purified, their genes and their promoters were sequenced, and their regulation was studied. The enzymes share homology (59% identity) but are easily distinguished by PAGE assay. Biochemical characterization, Edman degradation, and mass spectrometry demonstrated that EXC1Y and EXC2Y are both active as homodimers. Both genes are up-regulated by glucosamine (GlcN), in contrast to two endochitinases of this fungus. GlcN induces the secretion of several proteins (including a -glucosidase), among which EXC1Y is the most abundant. An exc2y knockout was constructed, to study the regulation of EXC1Y expression and secretion. The fungus has the ability to store a high amount of this enzyme in an active form and secrete it into the medium later.Communicated by J. Heitman     

Characterization of a versatile rhizospheric organism from cucumber identified as Ochrobactrum haematophilum

Several rhizobacteria play a vital role in promoting plant growth and protecting plants against fungal diseases and degrading pesticides in the environment. In this study, a bacterial strain, designated H10, was isolated from the rhizosphere at Laixi in Shandong Province, China, and was identified as Ochrobactrum haematophilum based on API 20 NE tests and 16S rRNA gene sequence analysis. The plant growth‐promoting characteristics of the strain were further characterized, and the results showed that strain H10 produces siderophore, indol‐3‐acetic (IAA) and solubilized phosphate but lacks 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity. Inoculation with the strain was found to significantly increase (p < 0.05) the growth of cucumber in pot experiments. Strain H10 was assessed in vitro for antagonism against several pathogenic fungi and showed high antifungal activity. The cell‐free culture filtrates, which had high extracellular chitinase, β;‐1,3‐glucanase and protease activities, could inhibit the growth of all pathogenic fungi tested, indicating that growth suppression was partly due to extracellular antifungal metabolites present in the culture filtrates. Changes in hyphal morphology were observed in phytopathogenic fungi after treatment with the culture filtrates. Additionally, strain H10 was able to degrade 80%, 85% and 58% of the pesticides chlorpyrifos, β;‐cypermethrin and imidacloprid, respectively, within 60 h in liquid culture. The inoculation of strain H10 into soil treated with 100 mg kg^–1of the three pesticides accordingly resulted in a higher degradation rate than in noninoculated soils. These results highlight the potential of this bacterium for use as a biofertilizer and biopesticide and suggest that it may provide an alternative to the use of chemical fertilizers and pesticides in agriculture. Additionally, it may represent a bioremediation agent that can remove contaminating chemical pesticide residues from the environment. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phylogenetic analysis of the Aspergillus niger aggregate in relation to feruloyl esterase activity

Species of the Aspergillus niger aggregate are known to produce feruloyl esterases, enzymes involved in the degradation of cell wall polymers. However, species delineation is difficult in these fungi. We combined AFLP analysis with ITS rDNA and beta-tubulin sequencing to characterize the isolates of this aggregate in terms of feruloyl esterase production. A preliminary re-examination of isolates based on comparison of ITS rDNA and beta-tubulin sequences with those of typical taxa deposited in international collections led us to re-identify the isolates as members of the species A. niger, A. foetidus and A. tubingensis. Molecular clustering based on beta-tubulin data and AFLP analysis showed that the strains of A. niger formed a homogenous phylogenetic group distinguished by either zero or type A feruloyl esterase activity, while strains A. foetidus and A. tubingensis exhibited type B feruloyl esterase activity when grown on sugar beet pulp.     

Putative use of a Bacillus subtilis L194 strain for biocontrol of Phoma medicaginis in Medicago truncatula seedlings

An antagonist L194 strain against Phoma medicaginis pathogenic fungi was isolated from Tunisian soil (vicinity of Tunis) and identified as Bacillus subtilis based on biochemical characteristics and partial 16S rDNA sequence. When cells were grown in a minimal medium for 24 h, spore culture supernatant exhibited 2-fold higher antifungal activity than vegetative cells. MALDI-TOF mass spectrometry analysis showed that L194 spores produced mainly iturins, surfactins and fengycins with long-chain fatty acids, and other not yet identified compounds. Both vegetative cells and spores of L194 efficiently reduced germination of P. medicaginis conidia. As revealed by atomic force microscopy, L194 spores modified conidia morphology from a regular to a deflated shape. Data suggest that lipopeptides interacted with the cytoplasmic membrane, causing pore formation. In vivo, L194 spores were highly protective against P. medicaginis by reducing disease symptoms and alleviating growth disturbance of Medicago truncatula seedlings. As a whole, the lipopeptide-producing L194 strain may be successfully used in biocontrol of plant diseases induced by phytopathogenic fungi such as P. medicaginis.     

Bacteriocin-like activity of Bacteroides fragilis group isolated from marmosets.

The ability of strains of the B. fragilis group, isolated from the oral cavity and intestine of marmosets, to produce bacteriorin-like substances in solid medium, in terms of auto-, iso- and heteroantagonism, was evaluated. Antagonistic activity was exhibited by 52% of the intestinal strains, 3 of which showed autoantagonistic activity. Three out of 9 oral strains isolated, tested against themselves, showed simultaneous isoantagonism to 4 indicator strains; but not autoantagonism. The same 9 oral strains, when tested against 16 reference strains, revealed interspecific activity only against 2 Gram-positive microorganisms. Higher activity, evaluated by the size of the inhibition halo, was observed in BHI-S agar, and greatest inhibition was obtained after 72 h of incubation.