Antimicrobial activity of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains

A bacterial strain with a high level of antimicrobial activity was isolated from soil and identified as Bacillus megaterium. Production of antibiotics by nine strains of this species from the collection of the State Research Institute for Genetics and Selection of Industrial Microorganisms was investigated. In submerged cultures, nine out of ten B. megaterium strains were found to produce antibacterial antibiotics differing in their spectra of action. Physicochemical characteristics of five compounds were described. Three of them belonged to peptide antibiotics. All five compounds were active against the methicillin-resistant strain Staphylococcus aureus INA 00761. Three of them were shown to be the previously undescribed compounds. Antibiotics produced by various B. megaterium strains were also active against the Leuconostoc mesenteroides VKPM B-4177 strain resistant to glycopeptide antibiotics and against gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922.     

Antagonistic and antimicrobial activities of some bacterial isolates collected from soil samples

Thirty seven bacterial cultures isolated from soil samples obtained from different locations were tested for their antagonistic activity against some fungal pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani, causal agents of collar rot of sunflower, wilts and root rots, respectively. Among them, 5 bacterial strains, viz., A1 6 (Bacillus sphaericus), K1 24 (Pseudomonas fluorescens), M1 42 (Bacillus circulans), M1 66 (Bacillus brevis) and T1 22 (Bacillus brevis) showed positive antagonistic activity. M1 66 was the most effective in inhibiting mycelial growth of S. rolfsii in vitro followed by M1 42, T1 22, K1 24 and A1 6. Only one bacterial strain i.e. M1 42 exhibited antagonistic activity against F. oxysporum, and none of the bacterial strains gave positive activity against R. solani. Furthermore, antimicrobial activities of all the 5 strains were checked against different test organisms. These strains showed their extensive inhibition effect particularly against gram-positive test bacteria (Staphylococcus aureus and Bacillus subtilis) and the test fungal strain (Candida albicans). On the other hand, B. brevis M1 66 and B. brevis T1 22 strains had an inhibitory effect against gram positive and gram-negative test bacteria (Escherichia coli and Proteus vulgaris) as well as the test fungal strain.     

Increased enzymatic activities and levels of superoxide anion and phenolic compounds in cultures of basidiomycetes after temperature stress

Selected strains of basidiomycetes (Abortiporus biennis, Trametes versicolor and Cerrena unicolor) were shown to produce enhanced extracellular peroxidase (EP), superoxide dismutase (SOD) and laccase activities following the exposure of 10-day-old fungal cultures to separate high and low temperature stress. The stressful conditions also caused an increase in the concentrations of phenol compounds and superoxide anion radicals in these cultures. At first, peroxidase activity was observed at 12 hours from the moment of temperature stress application. Laccase activity appeared at 96 hours after the maximum levels of superoxide anion radicals (48 h) and SOD activity (36–72 h). The concentration of phenolic substances grew steadily during the period of cultivation. These relations between laccase, SOD and EP as well as superoxide radicals and phenol levels in the environment of ligninolytic fungi seems to be important in the course of the biosynthesis or biodegradation of lignin, as the consequence of adaptation of these basidiomycetes to environmental temperature conditions.     

Phenotypic and biological properties of two antagonist <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Bacillus subtilis KB-1111 and KB-1122 were studied to illustrate their phenotypic and biological properties. Comparison of KB-1111 with KB-1122 in morphology was carried out by microscopy and agar plate assays. Biological assay of the test strains showed that they may possess different physiological pathways from those of reference strain ATCC6501. The assessment of antagonism against the indicator fungi showed that both test strains had broad antifungal characteristics against eight phytopathogenic fungi. Of those fungal species, Magnaporthe grisea P131, Sclerotinia sclerotiorum, and F. oxysporium exhibited high sensitivity to the test strains.     

Efficient production of Al(OH)3‐immobilized laccase with a Heterobasidion annosum strain selected by microplate screening

Aims: Wild‐type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. Methods and Results: By using a newly developed 96‐well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l^?1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger‐scale liquid cultures. Aeration with a sparger lead to a 3·8‐fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l^?1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short‐range ordered Al(OH)₃ particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)₃‐adsorbed laccase was suitable for repeated decolourization of indigo carmine. Conclusions: Microplate cultivation allowed a large‐scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)₃ proved to be suitable as direct immobilization technique. Significance and Impact of the Study: The microplate screening method simplifies strain and medium development for laccase production. Two novel fungal strains suitable for laccase production were identified. Procedures for simple and efficient production of immobilized H. annosum laccase were established.     

Screening for dioxin-degrading basidiomycetes from temperate and tropical forests

A total of 500 strains of basidiomycetes isolated from temperate forests in Japan and 379 strains from tropical forests in Indonesia were subjected to a laboratory screening for dioxin-degrading ability. At first, about 200 fungal strains were selected by their ability to decolorize Remazol Brilliant Blue R dye as an indicator of ligninolytic activities. Next, for excluding the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long-term or short-term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the remaining amounts of this model compound. We chose Bjerkandera adusta strain VH57 as a promising new candidate for dioxin degradation, because it gave 40% difference in 2,7-DCDD levels between the two treatments after 30 days of exposure.     

Antifungal activity of some Discomycetes

The antifungal activity was investigated in culture filtrates of 131 strains (41 genera and 104 species) of Ascomycetes — Discomycetes by testing against 6 fungal species which causes diseases in man. The anti-fungal spectrum was established for a Ciboria rufo-fusca strain, the only one found to inhibit all test organisms. This strain was also active against several gram-positive and gram-negative bacteria.     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.     

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

Aims: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram‐positive and Gram‐negative bacteria, and were tested against mastitis isolates from dairy cows. Methods and Results: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell‐free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4‐methyl‐2‐oxovaleric acid sodium salt. Conclusions: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. Significance and Impact of the Study: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.     

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. Received: 22 March 1999 / Received revision: 12 July 1999 / Accepted: 1 August 1999     

The ubiquity of natural adsorbable organic halogen production among basidiomycetes

 Recently, several species of basidiomycetes were shown to produce de novo high concentrations of chloroaromatic metabolites. Since these lignocellulose-degrading fungi play a major role in the ecosphere, the purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes. A total of 191 fungal strains were monitored for adsorbable organic halogen (AOX) production when grown on defined liquid media. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. A low production of 0.1–0.5 mg AOX/l was observed among 25% of the strains, a moderate production of 0.5–5.0 mg AOX/l was observed among 16% of the strains and 9% of the strains produced high levels (5–67 mg AOX/l). The latter group was dominated by species belonging to the genera Hypholoma, Mycena and Bjerkandera, showing specific AOX productions in the range 1074–30893 mg AOX/kg dry weight of mycelial biomass. Many highly ecologically significant fungal species were identified among the moderate to high producers. These species were also able to produce AOX when cultivated on natural lignocellulosic substrates. Hypholoma fasciculare and Mycena metata respectively produced up to 132 mg and 193 mg AOX/kg dry weight of forest litter substrate in 6 weeks. Received: 5 October 1995/Received revision: 28 December 1995/Accepted: 12 February 1996     

Enzyme Immunoassay of Herbicide Decomposition by Soil and Wood Decay Fungi

The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicilliumsp. 13 and noncellulolytic ones Humicola lanuginosaspp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using the enzyme immune assay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. HyphomyceteMycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of the thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80–92% in 40 days. Mycelia steriliaINBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.     

Antifungal Effects of Bacilysin and Fengymycin from Bacillus subtilis F-29-3 A Comparison with Activities of Other Bacillus Antibiotics

Of the two antifungal antibiotics produced by Bacillus subtilis F-29-3, the dipeptide compound bacilysin inhibits yeasts (and bacteria), whereas the formerly unknown fengymycin, a complex of closely related lipopeptide components, shows antibiotic activity against filamentous fungi. Bacilysin production, formerly known for a few strains only, could be demonstrated for all 12 wild-type cultures of Bacillus subtilis tested during this study. The antibiotic also occurs in some strains of three other Bacillus species considered as closely realted to B. subtilis. Members of the lipopeptide class of antifungal Bacillus metabolites were formed by 8 of 12 Bacillus subtilis-isolates and several other Bacillus strains. The antibiotics of F-29-3 were compared with antifungal metabolites of other Bacillus isolates using TLC, agar-diffusion techniques and tests demonstrating the capacity of six lipopeptide and peptide preparations to protect rice seedlings from phytomycosis due to Rhizoctonia solani. Fengymycin proved to be different from the other compounds tested. It was less toxic to the test plants and protected them better from Rhizoctonia disease than the other antibiotics of the study did.     

Selection against hypermutability in Escherichia coli during long term evolution

Summary A population of a mutT strain of E. coli was maintained in a chemostat for 2,200 generations. Afterwards the rate, of mutation to resistance to three antibiotics was determined by the Luria-Delbrück fluctuation test. It was found that the strain had a distinctly reduced mutability after the long-term cultivation compared with the original strain. Nevertheless the mutability was still much higher than that of a wild-type strain. After transduction of the mutT gene into another genetic backgroud the transductants showed the same mutability as the original strain indicating that the mutT allele itself had not changed. Our results support the hypothesis that under new environmental conditions mutator strains have an advantage due to their more efficient production of beneficial mutations. After optimal adaptation there is selection against high mutation rates due to the increased mutational load in the mutator population.     

Characterization of Enterobacteria Producing the Low-Molecular-Weight Antibiotics Microcins

A comparative study of the morphological, cultural, physiological, and biochemical properties of the microcinogenic strains EcS 5/98, EcS 6/98, and EcB 214/99 and the known microcin C51 producer Escherichia coli M17(p74) showed that these strains belong to the species E. coli. The strains produced microcins with molecular masses lower than 10 kDa. Microcin biosynthesis was stimulated by a deficiency of nutrients in the cultivation media. The microcins were found to be resistant to thermolysin but were degraded by pronase, protolichetrem, and the Bacillus mesentericus metalloproteinase. This indicated that the microcins are peptides or contain peptides in their molecules. The study of cross immunity to the microcins and the sequencing of their genetic determinants showed that the microcins of strains EcS 5/98 and EcS 6/98 are of B type, whereas the microcin of strain EcB 214/99 presumably belongs to another type, since it suppresses the growth of the producers of C and B-type microcins. The new microcin producers possess antibacterial activity against natural isolates belonging to the genera Escherichia and Salmonella, against a wide range of colicinogenic Escherichia strains, and against collection Salmonella cultures.     

Isolation and selection of novel basidiomycetes for decolorization of recalcitrant dyes

Thirty wood-rotting basidiomycetes, most of them causing white rot in wood, were isolated from fruiting bodies growing on decaying wood from the Sierra de Ayllón (Spain). The fungi were identified on the basis of their morphological characteristics and compared for their ability to decolorize Reactive Black 5 and Reactive Blue 38 (as model of azo and phthalocyanine type dyes, respectively) at 75 and 150 mg/L. Only eighteen fungal strains were able to grow on agar plates in the presence of the dyes and only three species (Calocera cornea, Lopharia spadicea, Polyporus alveolaris) decolorized efficiently both dyes at both concentrations. The ligninolytic activities, involved in decolorization dyes (laccases, lignin peroxidases, Mn-oxidizing peroxidases), were followed in glucose basal medium in the presence of enzyme inducers. The results indicate a high variability of the ligninolytic system within white-rot basidiomycetes. These fungal species and their enzymes can represent new alternatives for the study of new biological systems to degrade aromatic compounds causing environmental problems.     

Biochemical tests for identification of mycelial cultures of basidiomycetes

Twenty-four biochemical tests have been developed to identify basidiomycetes from mycelial cultures. Eleven are designated ‘instant tests’ because results are obtained within 24 h; ten of these test for specific enzymes, the other is based on a reaction between vanillin and unknown fungal metabolites. The remaining thirteen tests require incubation periods ranging from 1 to 4 days and are thus designated ‘incubated tests’. Sixteen of these tests were applied first to identified isolates of Peniophora sacrata, Sclerotium rolfsii, Stereum purpureum and Trametes versicolor. They distinguished these species satisfactorily, so these sixteen tests, and eight others devised subsequently, were applied to unidentified basidiomycetes isolated from diseased roots of fruit crops. The results of these tests enabled the isolates to be placed in seven groups. All isolates in a particular group were morphologically similar which further suggested that each group might correspond to a species and that isolates could be defined on the basis of biochemical tests.     

Removal of aluminium from aqueous solution by four wild-type strains of Aspergillus niger

This paper provides a unique comparison of the performance of four wild-type Aspergillus niger strains in remediation of aluminium(III)-contaminated aqueous solutions. The direct fungal aluminium removal via biosorption and bioaccumulation was compared among all fungal strains, including bioaccumulation efficiency during dynamic and static cultivation. Our results indicate that aluminium bioaccumulation by living biomass outperformed biosorption, although biosorption by non-living biomass is a less time-demanding process. Among others, only one strain significantly differed regarding comparison of dynamic and static bioaccumulation. In this case, a significantly higher removal performance was achieved under dynamic cultivation conditions at initial aluminium(III) concentrations over 2.5 mg L⁻¹. Although the fungal sensitivity towards aluminium(III) differed among selected fungal strains, there was no apparent correlation between the strains’ removal performance and their adaptive mechanisms.

     

天山大峡谷原始森林黏细菌的分离鉴定及其抗菌活性

【背景】黏细菌是一类具有多细胞群体行为特征的高等原核生物,其对植物病原真菌和细菌的捕食特性使其在植物病害防治方面具有重要的应用潜力。【目的】探究乌鲁木齐天山大峡谷原始森林可培养黏细菌的多样性并分析其抗菌活性,为发掘黏细菌生防菌株奠定基础。【方法】以天山大峡谷原始森林采集的土样和腐木为分离材料,采用兔粪诱导法和被捕食菌诱导法从中分离纯化黏细菌菌株,结合形态学观察、生理生化测定和16S rRNA基因序列分析确定其分类地位,并以6种植物病原真菌[大丽轮枝菌(Verticillium dahliae)、尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f. sp. vasinfectum)、拟轮枝链孢霉(Fusarium verticillioides)、立枯丝核菌(Rhizoctonia solani)、黄色镰刀菌(Fusarium culmorum)、细极链格孢菌(Alternaria tenuissima)]和1种植物病原细菌[梨火疫病菌(Erwinia amylovora)]为靶标菌,通过平板对峙法和菌苔捕食法测定其抗菌活性。【结果】从采集的样品中分离出70株菌株,经纯化后获得36株黏细菌纯培养物。经鉴定隶属于4个属,黏球菌属(Myxococcus) 30株、孢囊杆菌属(Cystobacter) 3株、珊瑚球菌属(Corallococcus) 2株和原囊菌属(Archangium) 1株。抗菌活性分析显示,本研究获得的36株黏细菌至少对2种植物病原真菌有抗菌活性,表现出广谱的抗真菌活性,初步筛选出一株菌株NSE37-1兼具广谱和高效抗真菌活性;供试的15株黏细菌对梨火疫病菌均具有捕食活性,初步筛选出一株对梨火疫病菌具有较强捕食能力的黏细菌菌株NSE25。【结论】天山大峡谷可培养黏细菌资源比较丰富,黏球菌属是该地区可培养黏细菌菌群中的优势菌。分离纯化出的黏细菌菌株均表现出广谱的抗植物病原菌活性,具有进一步研究和开发的潜在价值。     

白马雪山云南黄芪与灰毛康定黄芪根际微生物物种多样性及抗生物膜活性菌株筛选

【背景】细菌生物膜是造成病原菌耐药性增强和持续感染的主要因素,但目前尚无针对抗菌膜的特效药物。特境植物根际微生物可产生大量具有提高宿主免疫功能的活性成分,极具抗生物膜药源开发潜力。【目的】了解滇西北高寒特境白马雪山分布的云南黄芪与灰毛康定黄芪植物根际微生物的物种多样性,并对可培养菌株进行抑菌与抗生物膜活性筛选。【方法】采用宏基因组技术结合传统微生物培养方法,对采自我国云南迪庆藏族自治州德钦县白马雪山的云南黄芪与灰毛康定黄芪的根际微生物进行物种多样性研究,并通过“孔板法”测定其可培养菌株发酵液乙酸乙酯粗浸膏的抗菌、抗生物膜活性。【结果】宏基因组测序结果显示,云南黄芪根际土壤样本中的微生物来自6门7纲8目8科9属10种,其中栖热菌属为优势菌群;灰毛康定黄芪根际土壤样本中的微生物来自6门8纲10目11科14属15种,其中慢生根瘤菌属为优势菌群。通过纯培养共获得145株可培养菌株,包括112株细菌和33株真菌。其中,云南黄芪根际细菌59株,共计16属35种,优势属为假单胞菌属和链霉菌属;根际真菌19株,共计4属5种,优势属为曲霉属;灰毛康定黄芪根际细菌53株,归属于16属29种,优势属为芽孢杆菌属与寡养单胞菌属;根际真菌14株,归属于3属4种,优势属为曲霉属。从不同种水平上选择51株细菌和7株真菌为代表菌株进行抗生素药源评估,发现5株细菌及1株真菌发酵液的乙酸乙酯粗浸膏具有中等至较强的抗革兰阳性菌活性,而且其中4株具有抗MRSA生物膜活性,最终确定了链霉属放线菌Streptomyces fulvissimus KTA1和曲霉属真菌Aspergillus fumigatus YNF5为潜力活性菌株。【结论】首次报道了滇西北地区高寒特境黄芪属植物根际微生物具有较好的物种多样性,而且具有一定的抗生素药用资源开发潜力。本研究对滇西北高寒特境特色植物来源的微生物资源开发利用与保护具有重要的借鉴意义。