Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

Aims: To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria. Methods and Results: Eight Tunisian hydrocarbonoclastic oilfields bacteria have been isolated and selected for further characterization studies. Phylogenetic analysis revealed that three thermophilic strains belonged to the genera Geobacillus, Bacillus and Brevibacillus, and that five mesophilic strains belonged to the genera Pseudomonas, Lysinibacillus, Achromobacter and Halomonas. The bacterial strains were cultivated on crude oil as sole carbon and energy sources, in the presence of different NaCl concentrations (1, 5 and 10%, w/v), and at 37 or 55°C. The hydrocarbon biodegradation potential of each strain was quantified by GC–MS. Strain C450R, phylogenetically related to the species Pseudomonas aeruginosa, showed the maximum crude oil degradation potentiality. During the growth of strain C450R on crude oil (2%, v/v), the emulsifying activity (E24) and glycoside content increased and reached values of 77 and 1·33 g l^?1, respectively. In addition, the surface tension (ST) decreased from 68 to 35·1 mN m^?1, suggesting the production of a rhamnolipid biosurfactant. Crude biosurfactant had been partially purified and characterized. It showed interest stability against temperature and salinity increasing and important emulsifying activity against oils and hydrocarbons. Conclusions: The results of this study showed the presence of diverse aerobic bacteria in Tunisian oilfields including mesophilic, thermophilic and halotolerant strains with interesting aliphatic hydrocarbon degradation potentiality, mainly for the most biosurfactant produced strains. Significance and Impact of the Study: It may be suggested that the bacterial isolates are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon‐contaminated sites.     

New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential

Aims: To characterize bacilli isolated from shallow hydrothermal vents of Panarea Island (Italy) and evaluate their biotechnological potential. Methods and Results: Fifteen isolates were characterized by culture and molecular methods. Eleven isolates were thermophilic, six isolates were alkalophilic and four of them were haloalkalophilic. After 16S rRNA gene sequencing, four strains, exhibiting sequence similarity below 95% with deposited strains, may represent novel species of bacilli. One strain was strictly related to Geobacillus subterraneus, but shared phenotypic characteristics for which it could be considered a new strain of this species. Four strains were affiliated with different Bacillus spp. Most isolates produced gelatinase, lipases and amylase, and some were mercury tolerant. Exopolysaccharides (EPS) production was tested adding different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose, 1% w/v) as a carbon source in a minimal medium. The highest EPS yield (185 mg l^?1) was reached by strain 1A70 utilizing ribose as a carbon source. Conclusions: Novel strains of Geobacillus and indigenous ribotypes of Bacillus with biotechnological potential inhabit shallow vents of Panarea Island. Significance and Impact of the Study: New strains of thermophilic bacilli from Panarea are producers of useful biomolecules for industrial purposes as well as environmental and biotechnological applications.     

The Phylogenetic Diversity of Aerobic Organotrophic Bacteria from the Dagang High-Temperature Oil Field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagang high-temperature oil field (China), which is exploited with water-flooding, have been studied. Twenty-two strains of the most characteristic thermophilic and mesophilic aerobic organotrophic bacteria have been isolated from the oil stratum. It has been found that, in a laboratory, the mesophilic and thermophilic isolates grow in the temperature, pH, and salinity ranges characteristic of the injection well near-bottom zones or of the oil stratum, respectively, and assimilate a wide range of hydrocarbons, fatty acids, lower alcohols, and crude oil, thus exhibiting adaptation to the environment. Using comparative phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates has been established. The aerobic microbial community includes gram-positive bacteria with a high and low G+C content of DNA, and γ and β subclasses of Proteobacteria. The thermophilic bacteria belong to the genera Geobacillus and Thermoactinomyces, and the mesophilic strains belong to the genera Bacillus, Micrococcus, Cellulomonas, Pseudomonas, and Acinetobacter. The microbial community of the oil stratum is dominated by known species of the genus Geobacillus (G. subterraneus, G. stearothermophilus, and G. thermoglucosidasius) and a novel species “Geobacillus jurassicus.” A number of novel thermophilic oil-oxidizing bacilli have been isolated.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 401–409.Original Russian Text Copyright © 2005 by Nazina, Sokolova, Shestakova, Grigoryan, Mikhailova, Babich, Lysenko, Tourova, Poltaraus, Qingxian Feng, Fangtian Ni, Belyaev.     

Physiological and phylogenetic diversity of thermophilic spore-forming hydrocarbon-oxidizing bacteria from oil fields

The distribution and population density of aerobic hydrocarbon-oxidizing bacteria in the high-temperature oil fields of Western Siberia, Kazakhstan, and China were studied. Seven strains of aerobic thermophilic spore-forming bacteria were isolated from the oil fields and studied by microbiological and molecular biological methods. Based on the 16S rRNA gene sequences, phenotypic characteristics, and the results of DNA-DNA hybridization, the taxonomic affiliation of the isolates was tentatively established. The strains were assigned to the first and fifth subgroups of the genusBacillus on the phylogenetic branch of the gram-positive bacteria. Strains B and 421 were classified asB. licheniformis. Strains X and U, located betweenB. stearothermophilus andB. thermocatenulatus on the phylogenetic tree, and strains K, Sam, and 34, related but not identical toB. thermodenitrificans andB. thermoleovorans, undoubtedly represent two new species. Phylogenetically and metabolically related representatives of thermophilic bacilli were found to occur in geographically distant oil fields     

A refinery sludge deposition site: presence of nahH and alkJ genes and crude oil biodegradation ability of bacterial isolates

204 bacterial isolates from four Greek refinery sludge deposition sites were investigated for the presence of nahH and alkJ genes encoding key enzymes of both aromatic and aliphatic hydrocarbon degradation pathways by PCR and DNA hybridisation. Members of Pseudomonas, Acinetobacter, Bacillus, Rhodococcus and Arthrobacter play important role in bioremediation processes in sandy/loam soil contaminated with oil and nahH and alkJ genes were present in the 73% of the isolates. Consortia of bacterial isolates that were used for biodegradation of aliphatic and aromatic hydrocarbons in crude oil using liquid cultures exhibited rates from 35% to 48% within 10 days of incubation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Geobacillus galactosidasius sp. nov., a new thermophilic galactosidase-producing bacterium isolated from compost

Two thermophilic spore-forming strains, with optimum growth temperature at 70 °C, were isolated from compost of the “Experimental System of Composting” (Teora, Avellino, Italy). A phylogenetic analysis based on 16S rRNA gene sequences showed that these organisms represented a new species of the genus Geobacillus. Based on polyphasic taxonomic data the strains represented a novel species for which the name Geobacillus galactosidasius sp. nov. is proposed. The type strain is CF1B^T (= ATCC BAA-1450^T = DSM 18751^T).     

Phylogenetic diversity of thermophilic carbohydrate degrading bacilli from Bulgarian hot springs

Phylogenetic diversity of culturable bacteria from genus Bacillus and related genera, isolated from 18 Bulgarian hot springs was investigated in association with their functional diversity. Sixty-seven thermophilic and facultative thermophilic strains were isolated under aerobic conditions at 60°C. Sixty-six of them belonged to eight species in four genera from Bacillus group: Anoxybacillus, Geobacillus, Brevibacillus and Bacillus. Representatives of the genus Anoxybacillus predominated. Based on phylogenetic analysis (<97% sequence similarity) four strains belonged to groups representing potentially novel species. Producers of carbohydrases, degrading 12 from the tested 13 substrates were isolated. About half of the isolates degraded amylose by exo- or endo-mechanism of action of their enzymes. The isolates degrading hemicellulose carbohydrates like arabinan, arabinoxylan, β-glucan, galactan, galactomannan and xyloglucan were reached to. Some of the microorganisms were able to uptake microbial polysaccharides like curdlan and gellan and their enzymes were between first reported thermostable enzymes in their groups, like gellan lyase and curdlan lyase A relation between species affiliation and their functional activity was observed—all A. gonensis strains were producer of amylolytic enzymes, most of Brevibacillus ruber strains were able to grow in a minimal medium with xanthan.     

alkB homologs in thermophilic bacteria of the genus Geobacillus

Screening for alkane hydroxylase genes (alkB) was performed in thermophilic aerobic bacteria of the genus Geobacillus. Total DNAs were isolated from the biomass of 11 strains grown on a mixture of saturated C₁₀–C₂₀ hydrocarbons. Fragments of alkB genes were amplified by PCR with degenerate oligonucleotide primers, and the PCR products were cloned and sequenced. For the first time, a set of alkB gene homologs was detected in the genomes of thermophilic bacteria. The strains each contained three to six homologs, of which only two were common for all of the strains. Phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences showed that six of the variants revealed in Geobacillus were closely related to alkB4, alkB3, and alkB2, found in Rhodococcus erythropolis strains NRRL B-16531 and Q15. All variants of alkB sequences were unique. Analysis of the GC composition showed that the Geobacillus alkB homologs are closer to Rhodococcus than to Geobacillus chromosomal DNA. It was assumed that the alkB genes were introduced in the Geobacillus genome via interspecific horizontal transfer and that Rhodococcus or other representatives of Actinobacteria served as donors. Analysis of the codon usage in the fragments of alkB genes confirmed the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. The formation of a set of several alkB homologs in a genome of a particular microorganism may result from free gene exchange within this pool.     

Oil biodegradation by Bacillus strains isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil

Sixteen spore forming Gram-positive bacteria were isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil. These strains were identified as belonging to the genus Bacillus using classical biochemical techniques and API 50CH kits, and their identity was confirmed by sequencing of part of the 16S rRNA gene. All strains were tested for oil degradation ability in microplates using Arabian Light and Marlin oils and only seven strains showed positive results in both kinds of oils. They were also able to grow in the presence of carbazole, n-hexadecane and polyalphaolefin (PAO), but not in toluene, as the only carbon sources. The production of key enzymes involved with aromatic hydrocarbons biodegradation process by Bacillus strains (catechol 1,2-dioxygenase and catechol 2,3-dioxygenase) was verified spectrophotometrically by detection of cis,cis-muconic acid and 2-hydroxymuconic semialdehyde, and results indicated that the ortho ring cleavage pathway is preferential. Furthermore, polymerase chain reaction (PCR) products were obtained when the DNA of seven Bacillus strains were screened for the presence of catabolic genes encoding alkane monooxygenase, catechol 1,2-dioxygenase, and/or catechol 2,3-dioxygenase. This is the first study on Bacillus strains isolated from an oil reservoir in Brazil.     

Genetic Diversity among Thermophilic Bacteria Isolated from Geothermal Sites by Using Two PCR Typing Methods

Microbial communities thriving at two hot springs, Hammam Pharaon (Pharaoh's Bath) and Oyoun Mossa (Moses springs), in Egypt was studied by cultural and molecular methods. Thirteen morphologically distinct strains of facultative anaerobic thermophilic bacterial isolates have been characterized and identified using phenotypic and genotypic characters including RAPD-PCR, ERIC-PCR typing, plasmid analysis and 16S rRNA sequencing. All isolates produced plasmid DNA with various sizes ranging from 0.7 kb to a larger plasmid 7.2 kb. The bacterial strains could tolerate a temperature range between 45 to 85°C and a pH between 4–11. Also, sulphate-reducing bacteria (SRB) in the thermal springs were investigated with combined biochemical and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore-forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 30 to 50°C and could use pyruvate, lactate and ethanol as electron donors. The dissimilatory sulphite reductase (DSR) gene sequences of eleven representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris. 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Bacillus (one species), Anoxybacillus (four species) and Geobacillus (eight species). In this study phenotypic and genotypic diversity were applied for the first time to differentiate thermophilic bacteria of such geothermal sites in Sinai, Egypt.     

Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes

A thermophilic Bacillus strain NG80-2 growing within the temperature range of 45–73°C (optimum at 65°C) was isolated from a deep subterranean oil-reservoir in northern China. The strain was able to utilize crude oil and liquid paraffin as the sole carbon sources for growth, and the growth with crude oil was accompanied by the production of an unknown emulsifying agent. Further examination showed that NG80-2 degraded and utilized only long-chain (C15–C36) n-alkanes, but not short-chain (C8–C14) n-alkanes and those longer than C40. Based on phenotypic and phylogenic analyses, NG80-2 was identified as Geobacillus thermodenitrificans. The strain NG80-2 may be potentially used for oily-waste treatment at elevated temperature, a condition which greatly accelerates the biodegradation rate, and for microbial enhancing oil recovery process.Lei Wang, Yun Tang and Shuo Wang contributed equally to this study.     

基于可培养方法分析云南腾冲小空山火山谷芽胞杆菌分布特征

【目的】为了解云南腾冲小空山火山谷土壤中可培养芽胞杆菌种类分布特征。【方法】采用可培养手段对小空山火山谷阳坡、谷底和阴坡土壤中的芽胞杆菌进行分离培养,根据16S rRNA基因序列同源性对分离菌株进行鉴定,并分析系统发育地位。利用Canoco5软件分析采样点芽胞杆菌种类分布特征与土壤样品理化性质的相关性。【结果】从火山谷土壤样品中共分离获得180株芽胞杆菌,16S rRNA基因测序鉴定结果表明分离菌株隶属于芽胞杆菌纲2个科(芽胞杆菌科和类芽胞杆菌科)、6个属、34个种,其中芽胞杆菌属(Bacillus) 11个种,类芽胞杆菌属(Paenibacillus) 14个种,短芽胞杆菌属(Brevibacillus)3个种,赖氨酸芽胞杆菌属(Lysinibacillus)4个种,嗜冷芽胞杆菌属(Psychrobacillus)1个种和绿芽胞杆菌属(Viridibacillus)2个种,其中7个菌株与其最近模式菌株16SrRNA相似性低于种的界定阈值(98.65%),为芽胞杆菌潜在新物种。优势属为芽胞杆菌属和类芽胞杆菌属,优势种为蕈状芽胞杆菌(Bacillusmycoides),图瓦永芽胞杆菌(Bacillustoyonensis),蜡状芽胞杆菌(Bacilluscereus),解木糖赖氨酸芽胞杆菌(Lysinibacillusxylanilyticus),蜂房类芽胞杆菌(Paenibacillusalvei)和沙地绿芽胞杆菌(Viridibacillus arenosi)。其中16个种分离自阳坡,29个种分离自阴坡,9个种分离自谷底,三者共同种类为6种。阳坡、谷底和阴坡的芽胞杆菌种群分布Bray-Curtis相似性为62.4%,多样性分析结果表明,Shannon指数(H′)大小次序为阴坡阳坡谷底。环境因子分析发现,芽胞杆菌种群分布多样性特征与其土壤的海拔高度、碳氮比和硫含量呈负相关,而和碳源和氮源含量呈正相关。【结论】从以上结果得出,云南腾冲火山谷有着较为丰富的芽胞杆菌资源,且还存在可分离培养的芽胞杆菌的潜在新物种,为利用火山微生物资源提供了保障。     

Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species

The genus Geobacillus comprises thermophilic bacilli capable of endospore formation. The members of this genus provide thermostable proteins and can be used in whole cell applications at elevated temperatures; therefore, these organisms are of biotechnological importance. While these applications have been described in previous reviews, the present paper highlights the environmental adaptations and genome diversifications of Geobacillus spp. and their applications in evolutionary-protein engineering. Despite their obligate thermophilic properties, Geobacillus spp. are widely distributed in nature. Because several isolates demonstrate remarkable properties for cell reproduction in their respective niches, they seem to exist not only as endospores but also as vegetative cells in diverse environments. This suggests their excellence in environmental adaptation via genome diversification; in fact, evidence suggests that Geobacillus spp. were derived from Bacillus spp. while diversifying their genomes via horizontal gene transfer. Moreover, when subjected to an environmental stressor, Geobacillus spp. diversify their genomes using inductive mutations and transposable elements to produce derivative cells that are adaptive to the stressor. Notably, inductive mutations in Geobacillus spp. occur more rapidly and frequently than the stress-induced mutagenesis observed in other microorganisms. Owing to this, Geobacillus spp. can efficiently generate mutant genes coding for thermostable enzyme variants from the thermolabile enzyme genes under appropriate selection pressures. This phenomenon provides a new approach to generate thermostable enzymes, termed as thermoadaptation-directed enzyme evolution, thereby expanding the biotechnological potentials of Geobacillus spp. In this review, we have discussed this approach using successful examples and major challenges yet to be addressed.

     

Phylogenetic diversity of isolates belonging to genera Geobacillus and Aeribacillus isolated from different geothermal regions of Turkey

The phylogenetic diversity of 31 thermophilic bacilli belonging to genera Geobacillus and Aeribacillus were investigated which were isolated from various geothermal sites of Turkey. Twenty-seven of these isolates were found to be belonged within the genus Geobacillus, whereas 4 of them were identified as Aeribacillus pallidus. The comparative 16S rRNA gene sequence analyses revealed that the A. pallidus isolates displayed sequence similarity values from 98.0 to 99.6% to their closest relative. Furthermore, Geobacillus isolates showed sequence similarity values from 88.9 to 99.8% with the reference type strains. According to the phylogenetic analysis, isolates belonging to genus Geobacillus were diverged into nine clusters and among these isolates, 19 of them were identified as strains related to G. caldoproteolyticus, G. thermodenitrificans, G. stearothermophilus, G. thermoglucosidasius and G. toebii with the most abundant 13 isolates from G. caldoproteolyticus. Four of the Geobacillus isolates were named as unidentified mix group, as they found to be genetically very homogenous like their closely related type species: G. thermoleovorans, G. vulcani, G. lituanicus, G. kaustophilus, G. caldovelox, G. caldotenax, and G. uralicus. Moreover, the sequence comparisons of E173a, E265, C161ab and A142 isolates demonstrated that they represented novel species among genus Geobacillus as they shared lower than 96.7% sequence similarity to all the described type species. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. By ARDRA results, the isolates were able to be differentiated and clustered, the discriminative restriction fragments of these isolates and type species were determined and the novelty of E173, E265, C161ab and A142 isolates could be displayed. Some differentiating phenotypic characters and the ability of amylase, glucosidase and protease production of these bacilli were also studied and biotechnologically valuable thermostable enzyme producing isolates were introduced in order to use in further studies.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50–80°C and pH 6.0–8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA–DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542^T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.     

Utilization of a polyphasic approach in the taxonomic reassessment of antibiotic- and enzyme-producing <Emphasis Type="Italic">Bacillus</Emphasis> spp. isolated from the Philippines

Summary The taxonomy of 58 locally isolated antibiotic- and enzyme-producing Bacillus isolates deposited at the Philippine National Collection of Microorganisms (PNCM)-BIOTECH was reassessed in this study using a polyphasic approach since they had been only partially identified prior to deposition in the culture collection. The isolates had 41.1–69% G + C, and possessed the characteristic diaminopimelic acid (DAP) and fatty acid methyl ester (FAMEs) properties of Bacillus species. Molecular analysis using specific PCR primers differentiated the isolates into two major groups, the Bacillus cereus group and the Bacillus subtilis group. To further differentiate these isolates, they were subjected to 39 phenotypic tests. Using the dichotomous key constructed for Bacillus, 45 isolates maintained their original identities, five were named at the species level, and 12 were re-identified and renamed. These results showed that the classical phenotypic tests allowed the reclassification of the isolates, while modern techniques of chemotaxonomy and the molecular approach led only to genus and cluster classification and confirmation.     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.