Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Differentiation of Three Varieties of Rhizoctonia circinata; var. circinata, var. oryzae and var. zeae on the Basis of Cellular Fatty Acid Composition

Cellular fatty acid analysis was employed to differentiate three varieties of Rhizoctonia circinata ; var. circinata , var. oryzae and var. zeae . Eight fatty acids including myristic (14 : 0), pentadecanoic (15 : 0), palmitic (16 : 0), palmitoleic (16 : 1  cis 9), stearic (18 : 0), oleic (18 : 1  cis 9), linoleic (18 : 2  cis 9,12) and linolenic (18 : 3  cis 9,12) acids were present in isolates of all three varieties of R. circinata . Heptadecanoic acid (17 : 0) was detected in isolates of R. circinata var. zeae but not in isolates of R. circinata var. circinata or R. circinata var. oryzae . Palmitic, oleic and linoleic acids were the major fatty acids found, comprising 94–98% of the whole-cell fatty acid content. The remaining fatty acids were present in small amounts. Based on the composition (%) of fatty acids, isolates of R. circinata var. circinata , R. circinata var. oryzae and R. circinata var. zeae were clearly differentiated into three groups as shown by principal component and cluster analyses. This finding agrees well with the grouping of R. circinata into three varieties based on differences in colony morphology of the vegetative state. In principal component and cluster analysis, isolates of R. circinata var. circinata from Japan and Alaska were indistinguishable.     

Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Chemotaxonomic characterization of rice seedling blight complex using fatty acid methyl ester (FAME) profiles

Rice seedling blight is an important disease caused by a complex of fungi that include Fusarium, Rhizopus, Pythium, and Trichoderma species. A modified MIDI method was used for extraction of fatty acids from these causal pathogens, and fatty acid methyl ester (FAME) profiles were characterized. Factors that might affect fatty acid production, such as period of culture and saponification in extraction, were also evaluated. A total of 14 fatty acids were detected, and FAME profiles showed quantitative and qualitative variations by discriminant analysis and principal component analysis. Genus-specific FAME profiles consisting of the types of fatty acid produced and remarkable components of individual fatty acids were observed. The possibility of application as chemotaxonomic methods based on the FAME profiles for diagnosis of the rice seedling blight complex is also discussed.     

Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing

Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.     

诺卡氏菌型放线菌细胞中脂肪酸的气相色谱分析

采用Sherolock全自动微生物鉴定系统,用气相色谱法,分析测定了4株诺卡氏菌型放线菌分离菌株的脂肪酸成分和含量,结果表明,该法具有分辨率高,稳定、重现性好,简便易行等特点,在一定程度上与16S rRNA基因序列比较结果相一致,能在种及菌株水平上反映出放线菌的基因型,系统发育和分类关系,是一种较好的脂肪酸定量测定方法,尤其适用于分析大量的菌株或分离株,可应用于放线菌种水平的分类和快速鉴定.     

Comparison of signature lipid methods to determine microbial community structure in compost

The microbial community structure changes substantially during the composting process and simple methods to follow these changes can potentially be used to estimate compost maturity. In this study, two such methods, the microbial identification (MIDI) method and the ester-linked (EL) procedure to determine the composition of long-chain fatty acids, were applied to compost samples of different age. The ability of the two methods to describe the microbial succession was evaluated by comparison with phospholipid fatty acid (PLFA) analysis on the same samples.Samples were taken from a 200-l laboratory compost reactor, treating source-separated organic household waste. During the initial stages of the process, the total concentration of fatty acids in compost samples treated with the EL and MIDI methods was many times higher than with the PLFA method. This was probably due to the presence of fatty acids from the organic material in the original waste. However, this substantial difference between PLFA and the other two methods was not found later in composting. Although the PLFA method gave the most detailed information about the growth and overall succession of the microbial community, the much simpler MIDI and EL methods also successfully described the shift from the initially dominating straight chain fatty acids to iso- and anteiso branched, 10 Me branched and cyclopropane fatty acids in the later stages of the process. Thus, the MIDI and EL extraction methods appear to be suitable for analysis of microbial FAME profiles in compost, particularly in the later stages of the process.     

我国霍乱弧菌的脂肪酸分型研究

目的 对脂肪酸分型方法在霍乱弧菌菌株鉴定、菌株相似性分析等方面的应用价值进行评价。方法 选取了分离自我国的两个主要致病血清群的194株霍乱弧菌菌株（1961年以来的El Tor型和1992年以来的O139群）,提取脂肪酸,应用MIDI公司的脂肪酸分型系统,进行数据分析。结果 检测的所有菌株都含有的脂肪酸成分有13种。霍乱弧菌的判断符合率为88.6%。二维聚类分析没有成明显可区分的群, O139群霍乱弧菌的脂肪酸组成与O1群的相似,产毒与非产毒霍乱弧菌的脂肪酸成分没有显著差异。结论 脂肪酸分型对弧菌种的快速鉴定有应用价值,对霍乱弧菌的现场分离鉴定有辅助意义,在小样本暴发资料的研究中能够反映菌株之间的亲缘关系,但其对霍乱弧菌种属内的各种特征性菌群不具有鉴别能力。     

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.     

Characterisation,differentiation and biochemical diversity of Fusarium solani and Fusarium proliferatum based on cellular fatty acid profiles

The biochemical relationships between Fusarium solani and Fusarium proliferatum isolates were investigated using fatty acid analysis. Cellular fatty acid composition showed that palmitic acid, stearic acid, oleic acid and linoleic acid were the most abundant fatty acids in these species and accounted for 93.88 and 94.02% of the fatty acid profiles in F. solani and F. proliferatum, respectively. The most predominant fatty acids were linoleic acid (37.44%) in F. solani and oleic acid (39.81%) in F. proliferatum. The fatty acid compositions of F. solani and F. proliferatum were significantly different (p?<?0.05) for most of the individual fatty acids. This study demonstrated that fatty acid profiles may be useful to characterise and differentiate F. solani and F. proliferatum isolates at the species level. Using fatty acid analysis, biochemical diversity was observed among isolates of these species. The dendrogramme revealed that F. solani and F. proliferatum formed two distinct clusters with a distance of 7.2. Isolates of each species were clustered with each other, having a Euclidean distance of 6 and 6.6 for F. solani and F. proliferatum, respectively.     

Distinction of Prevotella intermedia and Prevotella nigrescens from endodontic bacteremia through their fatty acid contents

Prevotella nigrescens has recently been recognized as a new species distinct from Prevotella intermedia. The distinction is based largely on DNA-DNA hybridization, electrophoretic migration of malate and glutamate dehydrogenase, and peptidase and lipase activities of type strains. Gas chromatography of cellular fatty acids can be a useful adjunct for characterization and identification of bacterial species. In the present study, cellular fatty acid profiles were determined for seven strains of P. intermedia and six strains of P. nigrescens. Six of these 13 strains were isolated from the root canal and blood of three patients during endodontic therapy of teeth with Asymptomatic apical periodontitis. The bacteria were cultivated anaerobically in 10 mL prereduced anaerobically sterilized peptone-yeast extract-glucose broth for 24 h. Dried cells of each isolate were methanolysed and their fatty acid contents determined by the Microbial Identification System software package by MIDI. The data were treated by principal component analysis, which distinguished P. nigrescensfromP. intermedia. Cellular fatty acid profiles of these strains of the species in blood matched the profiles of their respective root canal isolates, as demonstrated by Euclidean Distance Square assessment. This suggested that the organisms in the root canal had spread to the bloodstream during endodontic treatment.     

Presence of double-stranded RNA and virus-like particles in Rhizopus isolates

Fungal isolates belonging to four Rhizopus species were screened for the presence of double-stranded RNA (dsRNA) molecules. Five (two R. stolonifer, two R. microsporus, and one R. oryzae) of the 27 isolates examined harboured such genetic elements. Electrophoresis of the nucleic acids revealed five RNA patterns, with 1-5 discrete dsRNA bands. The molecular sizes corresponding to these bands were 2.2-14.8 kb. Gel electrophoresis of purified virus-like particles (VLPs) indicated only one capsid of similar size in all virus-harbouring strains; when investigated by electron microscopy, they were found to be polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates an uncapsidated large dsRNA segment (14.8 kb) was observed. No phenotypic differences were observed between uninfected and virus-harbouring Rhizopus isolates.     

Dereplication of<Emphasis Type="Italic"> Streptomyces</Emphasis> soil isolates and detection of specific biosynthetic genes using an automated ribotyping instrument

The discrimination of distinct cultures among morphologically similar Streptomyces soil isolates (dereplication) and the detection of specific biosynthetic pathways in these strains are important steps in the selection of microorganisms to include in a natural products library. We have developed methods for analysis of actinomycetes using the RiboPrinter microbial characterization system, an automated instrument that performs ribotyping on bacterial samples. To evaluate our dereplication method, 26 Streptomyces isolates, obtained from soil samples collected in Maui, Hawaii, were ribotyped and compared with each other, using the RiboPrinter. The strains were also compared by 16S rDNA sequence analysis, MIDI fatty acid analysis, and LC-MS profiling of fermentation extracts. The RiboPrinter was able to identify closely related isolates and to discriminate between morphologically similar isolates with unique genetic, fatty acid and fermentation profiles. For the detection of biosynthetic genes, a 1,006-bp probe containing a portion of an adenylation domain of a non-ribosomal peptide synthetase (NRPS) was employed. Using this alternate probe in place of the standard ribosomal probe, the RiboPrinter was able to detect NRPS genes in several strains of Streptomyces. These results demonstrate that the RiboPrinter has multiple applications in a natural products research program.     

Changes in lipid, fatty acids and phospholipids composition of whole rice bran after solid-state fungal fermentation

The aim of this study was to evaluate fermented rice bran phospholipids, lipids and fatty acid content in a fermentation solid system with Rhizopus oryzae fungus. For this, aliquots were withdrawn every 24h over 120 h. The content of phospholipids was determined by colorimetric method. Esterified fatty acids were separated by gas chromatography, then identified and quantified. The total lipids from fermented rice bran (FB) decreased from 20.4% to 11.2% in the range between 0 h and 120 h of fermentation while phospholipid contents were increased up to 2.4 mg P g(lipid)(-1). In fermented bran, oleic, palmitic and linoleic acids prevailed, with a decrease in saturated fatty acids (20%) and increase in the unsaturated ones (5%). This study showed that rice bran fermentation with R. oryzae can be applied to the production of phospholipids altering the saturated to unsaturated fatty acid ratio.     

Accuracy, Reproducibility, and Interpretation of Fatty Acid Methyl Ester Profiles of Model Bacterial Communities

We determined the accuracy and reproducibility of whole-community fatty acid methyl ester (FAME) analysis with two model bacterial communities differing in composition by using the Microbial ID, Inc. (MIDI), system. The biomass, taxonomic structure, and expected MIDI-FAME profiles under a variety of environmental conditions were known for these model communities a priori. Not all members of each community could be detected in the composite profile because of lack of fatty acid “signatures” in some isolates or because of variations (approximately fivefold) in fatty acid yield across taxa. MIDI-FAME profiles of replicate subsamples of a given community were similar in terms of fatty acid yield per unit of community dry weight and relative proportions of specific fatty acids. Principal-components analysis (PCA) of MIDI-FAME profiles resulted in a clear separation of the two different communities and a clustering of replicates of each community from two separate experiments on the first PCA axis. The first PCA axis accounted for 57.1% of the variance in the data and was correlated with fatty acids that varied significantly between communities and reflected the underlying community taxonomic structure. On the basis of our data, community fatty acid profiles can be used to assess the relative similarities and differences of microbial communities that differ in taxonomic composition. However, detailed interpretation of community fatty acid profiles in terms of biomass or community taxonomic composition must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.     

Comparison of Whole-Cell Fatty Acid (MIDI) or Phospholipid Fatty Acid (PLFA) Extractants as Biomarkers to Profile Soil Microbial Communities

The whole-cell lipid extraction to profile microbial communities on soils using fatty acid (FA) biomarkers is commonly done with the two extractants associated with the phospholipid fatty acid (PLFA) or Microbial IDentification Inc. (MIDI) methods. These extractants have very different chemistry and lipid separation procedures, but often shown a similar ability to discriminate soils from various management and vegetation systems. However, the mechanism and the chemistry of the exact suite of FAs extracted by these two methods are poorly understood. Therefore, the objective was to qualitatively and quantitatively compare the MIDI and PLFA microbial profiling methods for detecting microbial community shifts due to soil type or management. Twenty-nine soil samples were collected from a wide range of soil types across Oregon and extracted FAs by each method were analyzed by gas chromatography (GC) and GC-mass spectrometry. Unlike PLFA profiles, which were highly related to microbial FAs, the overall MIDI-FA profiles were highly related to the plant-derived FAs. Plant-associated compounds were quantitatively related to particulate organic matter (POM) and qualitatively related to the standing vegetation at sampling. These FAs were negatively correlated to respiration rate normalized to POM (Resp_POM), which increased in systems under more intensive management. A strong negative correlation was found between MIDI-FA to PLFA ratios and total organic carbon (TOC). When the reagents used in MIDI procedure were tested for the limited recovery of MIDI-FAs from soil with high organic matter, the recovery of MIDI-FA microbial signatures sharply decreased with increasing ratios of soil to extractant. Hence, the MIDI method should be used with great caution for interpreting changes in FA profiles due to shifts in microbial communities.     

Fatty acid analysis as a chemotaxonomic tool for taxonomic and epidemiological characterization of four fish pathogenic Tenacibaculum species

Aims: In this work, fatty acid content and profiles were analysed in order to differentiate the species Tenacibaculum maritimum, Tenacibaculum gallaicum, Tenacibaculum discolor and Tenacibaculum ovolyticum that are pathogenic for cultured marine fish and to assess the potential of fatty acid profiles as a tool for epizootiological typing. Methods and Results: The fatty acid methylesters (FAMEs) were extracted from cells grown on marine agar for 48 h at 25°C and were prepared and analysed according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System. The cellular fatty acid profiles of Tenacibaculum strains tested were characterized by the presence of large amounts of branched (36·1–40·2%) and hydroxylated (29·6–31·7%) fatty acids. The FAME products from the four species significantly (P < 0·05) differed in the content of iso‐C_15:03‐OH, iso‐C_16:03‐OH, iso‐C_15:1G, summed feature 3 (a component that contains C_16:1ω7c and/or iso‐C_15:0 2‐OH), iso‐C_16:0, C_17:1ω6c, C_15:03‐OH, iso‐C_17:03‐OH. Conclusions: Results of present study demonstrated the existence of differences in the fatty acids content between the T. maritimum isolates from different marine fish/geographical origin and between strains of T. maritimum, T. discolor, T. gallaicum and T. ovolyticum. Significance and Impact of the Study: Profiling of fatty acids may be a useful tool to distinguish T. maritimum from other Tenacibaculum species pathogenic for fish as well as for epizootiological differentiation of T. maritimum isolates.     

Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism

The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.     

Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate.